Protein s antibodies, methods of making and uses thereof

ABSTRACT

Provided here are antibodies that bind Protein S, and methods of making and using such antibodies. In some embodiments, the Protein S antibodies provided herein are useful for treating a bleeding disorder or platelet disorder, or a condition characterized by reduced or impaired blood coagulation and/or clotting, e.g. by depleting the circulating levels of TFPI, by disrupting a Protein S-TFPI complex, and/or by preventing the formation of a Protein S-TFPI complex.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Pat. Application No. 63/020,505, filed on May 5, 2020 and to U.S. Provisional Pat. Application No. 63/169,755 filed Apr. 1, 2021. The contents of each of the preceding applications are incorporated by reference herein in their entirety.

BACKGROUND

Protein S (also known as ProS, ProS1) is a vitamin K-dependent plasma protein involved in the coagulation cascade. The protein is multi-modular, comprising a γ-carboxy-glutamic acid domain (Gla domain), an epidermal growth factor-like domain (EGF domain), a thrombin-sensitive region (TSR), and a sex hormone binding globulin-like domain (SHBG-like domain). The protein is found in both a free form and as part of a complex with proteins such as C4 binding protein (C4BP) and tissue factor pathway inhibitor (TFPI). Protein S and TFPI are believed to exist as a complex in circulation. Among other functions, in its free form, Protein S is a cofactor in at least two pathways of the coagulation cascade: (1) Protein S is a cofactor for plasma activated Protein C (APC), involved in the inactivation and degradation of coagulation factors Factor Va and Factor VIIIa; and (2) Protein S is also a cofactor for TFPI, also present in plasma, involved in the inactivation of coagulation factors Factor Xa and Factor VIIa.

As Protein S and TFPI are bound together in the circulation and act together in down-regulating thrombin formation and coagulation, Protein S is a potential therapeutic target, useful for the treatment of bleeding disorders. Thus there is a need for agents that bind and modulate its activities within the coagulation pathway.

SUMMARY

Provided here are antibodies that bind Protein S, and methods of making and using such antibodies. In some embodiments, the Protein S antibodies provided herein are useful for treating a bleeding disorder, or a condition characterized by reduced or impaired blood coagulation and/or clotting.

Accordingly, in one aspect, provided herein are antibodies that bind Protein S, wherein the binding of the antibody to Protein S decreases circulating TFPI levels.

In another aspect, provided herein are antibodies that bind Protein S, wherein the binding of the antibody to Protein S disrupts a Protein S-TFPI complex, or prevents the formation of a Protein S-TFPI complex.

In another aspect, provided herein are antibodies that bind Protein S, wherein the antibodies are inhibitors of the cofactor activity of Protein S for TFPI.

In another aspect, provided herein are antibodies that bind Protein S, wherein the antibodies are capable of promoting coagulation and/or modulating a component in the coagulation cascade.

In another aspect, provided herein are exemplary Protein S antibodies comprising any one or more of the amino acid sequences of the complementarity determining region (CDR) sequences provided in Tables 1A, 1B, 1C, 2A, 2B, and 2C. In another aspect, the exemplary Protein S antibodies comprise any one of the CDR-L1 amino acid sequences of Table 1A; any one of the CDR-L2 amino acid sequences of Table 1B; any one of the CDR-L3 amino acid sequences of Table 1C; any one of the CDR-H1 amino acid sequences of Table 2A; any one of the CDR-H2 amino acid sequences of Table 2B; and any one of the CDR-H3 amino acid sequences of Table 2B. In another aspect, provided herein are exemplary Protein S antibodies comprising the numbered combinations of variable light chains and variable heavy chains presented in Table 4C. In another aspect, provided herein are the sequences of three different antibodies as presented in Table 6. In another aspect, provided herein are nucleic acids encoding for any of the Protein S antibodies provided herein.

In another aspect, provided herein is a pharmaceutical composition comprising any one of the Protein S antibodies provided herein, and optionally a pharmaceutically acceptable excipient.

Provided herein are methods of using the exemplary Protein S antibodies described herein. Accordingly, in one aspect, provided herein is an in vitro method of promoting the coagulation of blood, comprising contacting any one of the exemplary Protein S antibodies provided herein with a blood sample.

In another aspect, provided herein is a method of promoting the coagulation of blood in a subject in need thereof, comprising administering to the subject any of the exemplary Protein S antibodies provided herein, or pharmaceutical compositions provided herein. In exemplary embodiments, the route of administration is subcutaneous.

In another aspect, provided herein is a method of promoting the generation of thrombin in a subject in need thereof, comprising administering to the subject any of the exemplary Protein S antibodies provided herein, or pharmaceutical compositions provided herein.

In another aspect, provided herein is a method of treating a condition in a subject in need thereof, comprising administering to the subject any of the exemplary Protein S antibodies provided herein, or the pharmaceutical compositions provided herein, wherein the condition is selected from the group consisting of: bleeding disorders, platelet disorders, trauma, and bleeding resulting from a surgery, a medical procedure, and combinations thereof.

In another aspect, provided herein is the use of any one of the Protein S antibodies or pharmaceutical compositions provided herein, for the treatment of a condition in a subject in need thereof. The condition may be selected from the group consisting of: bleeding disorders, platelet disorders, trauma, bleeding resulting from a surgery or a medical procedure, and combinations thereof.

In another aspect, any one of the Protein S antibodies or pharmaceutical compositions provided herein, may be used for the manufacture of a medicament for the treatment of a condition in a subject in need thereof. The condition may be selected from the group consisting of: bleeding disorders, platelet disorders, trauma, bleeding resulting from a surgery or a medical procedure, and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic diagram of Protein S showing the modular domains of Protein S.

FIGS. 2A-2B depict the results of screening assays performed to assess APC and TFPI cofactor activity, respectively, using control Protein S antibodies. In this and subsequent figures, ProS = Protein S; mAb = monoclonal antibody; pAb = polyclonal antibody; PRP = platelet rich plasma.

FIGS. 3A-3G depict the characterization of Antibody 5 and Antibody 17, antibodies sharing the same human variable region, and are characterized as TFPI cofactor inhibitors.

FIGS. 4A-4E depict the characterization of Antibody 10 and Antibody 22, antibodies sharing the same human variable region, and are characterized as TFPI cofactor inhibitors.

FIG. 4F depicts the characterization of Antibody 26 as a TFPI cofactor inhibitor.

FIGS. 5A-5B depict the effect of selected Protein S antibodies on fibrin deposition.

FIGS. 6A-6B depict the effects of Antibody 5 in an in vitro microfluidics Hemophilia A bleeding model.

FIGS. 7A-7B depict the effect of Antibody 5 on enhanced dose-dependent thrombin generation, wherein the Protein S antibodies were added to plasma obtained from patients with various types of von Willebrand disease.

FIGS. 8A-8H depict the results of an ex vivo pharmacodynamic and pharmacokinetic assays run on samples taken from cynomolgus monkeys dosed with Protein S antibodies.

FIGS. 9A-9C depict the ability of the Protein S antibodies of the disclosure to prevent the formation of a Protein S-TFPI complex, by measuring the ability of TFPI to bind to a Protein S-Protein S Antibody complex. FIG. 9A depicts the results obtained with Antibody 5; FIG. 9B depicts the results obtained with Antibody 10; and FIG. 9C depicts the results obtained with a control antibody that does not prevent such formation.

FIG. 10 depicts the decreasing TFPI levels in plasma of cynomolgus monkeys over time, following repeated administration of Antibody 5, as assessed by ELISA.

FIG. 11 depicts the change in plasma TFPI levels observed after each antibody was dosed in cynomolgus monkeys at 3 mg/kg IV. The data show that dosing with Antibodies 5, 10, and 26 induced a drop in circulating TFPI levels.

FIGS. 12A-12B depict the effects of different concentrations of Antibody 5 and Antibody 10 in an in vitro thrombin generation assay using Protein S depleted human plasma. These assays were carried out to measure the effects of Antibody 5 on the prevention of formation of a TFPI-Protein S complex.

DETAILED DESCRIPTION

Provided herein are antibodies that bind Protein S, and methods of making and using such antibodies. Antibodies that bind to Protein S and inhibit its cofactor activity for TFPI, or disrupt its complexation with TFPI, or prevent its complexation with TFPI, may be useful in the treatment of bleeding disorders and other related diseases by promoting clot formation. Accordingly the Protein S antibodies may be useful for treating bleeding disorders. For example, the Protein S antibodies may be used for methods of treating conditions associated with or characterized by reduced blood clotting.

Where elements are presented in a list format (e.g., in a Markush group), it should be understood that each possible subgroup of the elements is also disclosed, and that any one or more elements can be removed from the list or group.

It should be understood that, unless clearly indicated, in any method described or disclosed herein that includes more than one act, the order of the acts is not necessarily limited to the order in which the acts of the method are recited, but the disclosure encompasses exemplary embodiments in which the order of the acts is so limited.

The terms used throughout the specification are defined as follows unless otherwise limited in specific instances. As used in the specification and the claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. All technical and scientific terms, acronyms, and abbreviates used in the specification and claims have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains, unless defined or stated otherwise. All numerical ranges are inclusive of the values defining the range as well as all integer values in between, unless indicated or defined otherwise.

The term “antibody” as used herein throughout is used in the broadest sense and may include a monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, non-human antibody, chimeric antibody, a monovalent antibody, an antigen-binding fragment (e.g., a Fab fragment, a Fab′2 fragment, an scFv), and other antibody fragments that retain specificity for and binding of Protein S. In some embodiments, the antibodies are monoclonal antibodies. In some embodiments, the antibodies are monoclonal antibodies. In some embodiments, the antibodies are monoclonal human antibodies. In some embodiments, the antibodies are monoclonal humanized antibodies. In some embodiments, the antibodies are monoclonal chimeric antibodies. In some embodiments, the antibodies contain a Fc domain.

Also provided herein are antibody-drug conjugates, bispecific antibodies, and multispecific antibodies that exhibit specificity for and binding of Protein S.

The terms “polynucleotide” and “nucleic acid” are used interchangeably herein, and refer to a polymeric form of nucleotides of any length, which may be ribonucleotides or deoxyribonucleotides. The terms include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms encompass nucleic acids containing known analogues of natural nucleotides and having similar binding properties, and are metabolized in a manner similar to naturally-occurring nucleotides, unless specifically limited or stated otherwise.

When a nucleic acid or amino acid sequence is said to have a certain percent “sequence identity” or “identity” or is a certain percent “identical” to another nucleic acid or amino acid sequence, that percentage of bases or amino acids are the same, and in the same relative position, when the sequences are aligned, when comparing the two sequences.

The term “subject,” as used herein refers to any subject for whom treatment or therapy is provided. The subject may be a mammalian subject. Mammalian subjects include, e. g., humans, non-human primates (e.g., cynomolgus monkey), rodents, (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, and the like), etc. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate, e.g. a cynomolgus monkey. In some embodiments, the subject is a companion animal (e.g. cats, dogs).

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

I. Antibodies That Bind and Modulate Protein S Activity A. Protein S Antibodies

Provided here are antibodies that bind to Protein S, and in some embodiments are specific for Protein S. The Protein S can be of any species, e.g. any mammalian species. In some embodiments, the Protein S antibody binds to human Protein S. In some embodiments, the Protein S antibody binds to the Protein S of non-human primates. In some embodiments, the non-human primate is cynomolgus monkey.

The amino acid sequence of human Protein S, targeted by antibodies of the disclosure, is shown below as SEQ ID. NO: 136.

10 20 30 40 50 MRVLGGRCGA LLACLLLVLP VSEANFLSKQ QASQVLVRKR RANSLLEETK           60 70 80 90 100 QGNLERECIE ELCNKEEARE VFENDPETDY FYPKYLVCLR SFQTGLFTAA          110 120 130 140 150 RQSTNAYPDL RSCVNAIPDQ CSPLPCNEDG YMSCKDGKAS FTCTCKPGWQ          160 170 180 190 200 GEKCEFDINE CKDPSNINGG CSQICDNTPG SYHCSCKNGF VMLSNKKDCK          210 220 230 240 250 DVDECSLKPS ICGTAVCKNI PGDFECECPE GYRYNLKSKS CEDIDECSEN          260 270 280 290 300 MCAQLCVNYP GGYTCYCDGK KGFKLAQDQK SCEVVSVCLP LNLDTKYELL          310 320 330 340 350 YLAEQFAGVV LYLKFRLPEI SRFSAEFDFR TYDSEGVILY AESIDHSAWL          360 370 380 390 400 LIALRGGKIE VQLKNEHTSK ITTGGDVINN GLWNMVSVEE LEHSISIKIA          410 420 430 440 450 KEAVMDINKP GPLFKPENGL LETKVYFAGF PRKVESELIK PINPRLDGCI          460 470 480 490 500 RSWNLMKQGA SGIKEIIQEK QNKHCLVTVE KGSYYPGSGI AQFHIDYNNV          510 520 530 540 550 SSAEGWHVNV TLNIRPSTGT GVMLALVSGN NTVPFAVSLV DSTSEKSQDI          560 570 580 590 600 LLSVENTVIY RIQALSLCSD QQSHLEFRVN RNNLELSTPL KIETISHEDL          610 620 630 640 650 QRQLAVLDKA MKAKVATYLG GLPDVPFSAT PVNAFYNGCM EVNINGVQLD          660 670 LDEAISKHND IRAHSCPSVW KKTKNS (SEQ ID. NO: 136)

The Protein S antibodies provided herein are capable of modulating one or more activities of Protein S upon binding, including, for example, reducing or inhibiting Protein S cofactor activity for TFPI selectively (interchangeably referred to herein as TFPI cofactor inhibitor, TFPI cofactor specific inhibitor, or TFPI pathway inhibitor), modulating the ability of Protein S to act as a carrier for TFPI (e.g. by disrupting a Protein S-TFPI complex, or by preventing the formation of a Protein S-TFPI complex), decreasing circulating TFPI levels, as well as promoting coagulation and altering levels of markers associated with coagulation, and modulating a component in the coagulation cascade.

TFPI is an inhibitor of procoagulant activity and is produced as at least two alternatively spliced isoforms in humans, TFPIα, and TFPIβ, which differ in domain structure and mechanism for cell surface association. TFPIα, but not TFPIβ contains Kunitz domain 3, the domain which is believed to be involved in binding to Protein S. Without being held to any theory or mechanism, it is believed that the Protein S antibodies of the disclosure inhibit the cofactor activity of Protein S for at least TFPIα, as it contains Kunitz domain 3.

In some embodiments, the Protein S antibodies provided herein can disrupt the interaction (complexation) between Protein S and TFPI. Protein S and TFPI exist in plasma as a protein complex, the formation of which may extend the half-life of TFPI in plasma. Without being bound to theory or mechanism, the disruption of the interaction between Protein S and TFPI by the Protein S antibodies provided herein may reduce the half-life of TFPI in plasma and lead to a decrease in TFPI levels. In some embodiments, the effect of the Protein S antibodies provided herein could be on preventing the formation of a TFPI-Protein S complex. Thus, in some embodiments, the administration of the Protein S antibodies provided herein to a subject in vivo results in lower TFPI levels in plasma.

In some embodiments, the Protein S antibodies provided herein are capable of modulating the Protein S ability to act as a cofactor within pathways of the coagulation cascade. In some embodiments, the Protein S antibodies provided herein are useful for reducing or inhibiting the cofactor activity of Protein S for TFPI (such antibodies are referred to herein as TFPI cofactor inhibitors (interchangeably referred to herein as TFPI cofactor specific inhibitors or TFPI pathway inhibitors). Such reduction in cofactor activity for TFPI leads to an increase in coagulation activity, as discussed herein. The antibodies provided herein show minimal or negligible APC cofactor inhibition.

In some embodiments, the Protein S antibodies provided herein are capable of decreasing the levels of circulating TFPI. For example, TFPI levels may be decreased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% as compared to in the absence of the Protein S antibodies. In some embodiments, the decrease in TFPI levels is accompanied by a concomitant decrease in Protein S levels. In other embodiments, the decrease in TFPI levels is not accompanied by a concomitant decrease in Protein S levels.

In some embodiments, the Protein S antibodies provided herein are capable of causing a reduction in the activity of TFPI. For example, TFPI activity may be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% as compared to in the absence of the Protein S antibodies.

In some embodiments, the antibodies are capable of promoting clotting of blood by reducing an ability of Protein S to act as a cofactor for TFPI, or reducing an ability of Protein S to act as a carrier for TFPI (either by disrupting Protein S-TFPI complexes, or by preventing the formation of Protein S-TFPI complexes). For example, clotting of blood may be promoted by reducing TFPI activity by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% as compared to in the absence of the Protein S antibodies.

In some embodiments, the Protein S antibodies provided herein are capable of promoting the generation of a marker associated with coagulation activity, and this can be exhibited in vitro (e.g. in a sample) and/or in vivo (e.g. upon administration to a subject). Such markers include, but are not limited to, thrombin, fibrin, D-dimer, clot formation, thrombin-antithrombin complex, fibrin degradation products, and prothrombin fragment F1.2.

In some embodiments, the Protein S antibodies provided herein are capable of promoting thrombin generation, restoring thrombin generation, or restoring the levels of thrombin, e.g., in a subject in need thereof, or in a sample. In some embodiments, the antibodies are capable of promoting generation of thrombin in a subject in need thereof. In some embodiments, the generation of thrombin does not exceed a predetermined threshold level. In some embodiments, the generation of thrombin is partially restored. In some embodiments, the generation of thrombin does not exceed a predetermined percentage of a maximum thrombin generation. In some embodiments, the generation of thrombin does not exceed a predetermined percentage of an area under the curve of the maximum thrombin generation. In some embodiments, the effect of the antibody on the generation of thrombin is antibody concentration-dependent or antibody dose-dependent. In some embodiments, thrombin generation may be increased by about 5-fold to 50-fold, e.g. by about 5-fold, by about 10-fold, by about 15-fold, by about 20-fold, by about 25-fold, by about 30-fold, by about 35-fold, by about 40-fold, by about 45-fold, or even by about 50-fold, as compared to in the absence of the Protein S antibodies.

In some embodiments, the Protein S antibodies provided herein can restore or promote thrombin generation in a subject who is deficient in coagulation factors (e.g. such as Factor VII deficiency, Factor VIII deficiency, Factor IX deficiency, Factor XI deficiency). In some exemplary embodiments, the coagulation factor deficiency is congenital. In some exemplary embodiments, the coagulation factor deficiency is acquired. In some embodiments, the Protein S antibodies provided herein can promote thrombin generation in a subject who is deficient in Factor VII, Factor VIII, Factor IX, Factor XI. For example, thrombin generation can be increased by about 5-fold to 50-fold, e.g. by about 5-fold, by about 10-fold, by about 15-fold, by about 20-fold, by about 25-fold, by about 30-fold, by about 35-fold, by about 40-fold, by about 45-fold, or even by about 50-fold, as compared to in the absence of the Protein S antibodies.

In some embodiments, the Protein S antibodies provided herein can promote thrombin generation in a subject who has von Willebrand disease (vWD). In some embodiments, the vWD is a subtype selected from: vWD Type 1, vWD Type 2A, vWD Type 2B, vWD Type 2N, vWD Type 2M, vWD Type 3, and acquired vWD. In some exemplary embodiments, the vWD is a subtype selected from Type 1, Type 2, or Type 3. For example, thrombin generation can be increased by about 5-fold to 50-fold, e.g. by about 5-fold, by about 10-fold, by about 15-fold, by about 20-fold, by about 25-fold, by about 30-fold, by about 35-fold, by about 40-fold, by about 45-fold, or even by about 50-fold, as compared to in the absence of the Protein S antibodies.

In some embodiments, the Protein S antibodies provided herein are capable of promoting fibrin generation, e.g., in a subject in need thereof, or in a sample. In some embodiments, the Protein S antibodies provided herein are capable of promoting fibrin deposition e.g., in a sample. In some embodiments, the Protein S antibodies provided herein are capable of promoting coagulation activity, wherein the coagulation activity is marked by a promotion of fibrin generation. For example, fibrin generation may be increased by 5-fold to 50-fold, e.g. by about 5-fold, by about 10-fold, by about 15-fold, by about 20-fold, by about 25-fold, by about 30-fold, by about 35-fold, by about 40-fold, by about 45-fold, or even by about 50-fold, as compared to in the absence of the Protein S antibodies, as compared to in the absence of the Protein S antibodies.

In some embodiments, the Protein S antibodies provided herein are capable of promoting increasing D-dimer levels, e.g., in a subject in need thereof, or in a sample. In some embodiments, the Protein S antibodies provided herein are capable of promoting in vivo coagulation activity, wherein the coagulation activity is marked by an increase in D-dimer levels upon administration of the Protein S antibodies. For example, D- dimer levels may be increased by about two-fold to about 1000-fold.

In some embodiments, the Protein S antibodies provided herein are capable of promoting coagulation in a sample or in a subject. In some embodiments, the Protein S antibodies provided herein alter the levels of markers associated with coagulation activity in a sample or in a subject. For example, in some embodiments, the antibodies are capable of restoring thrombin generation in a sample, or in a subject. In some embodiments, the antibodies are capable of restoring fibrin deposition in a sample, or in a subject. In some embodiments, the antibodies provided herein are capable of promoting a restoration of fibrin deposition. In some embodiments, the antibodies provided herein are capable of increasing the levels of D-dimer in a sample, or in a subject. In some embodiments, the antibodies provided herein are capable of promoting an increase of D-dimer. In some embodiments, activity of the antibodies provided herein is dose-dependent. In some embodiments, activity of the antibodies provided herein is measured in vitro. In some embodiments, activity of the antibodies provided herein is measured in vivo.

In some embodiments, provided herein are Protein S antibodies comprising a binding affinity (KD) to Protein S of about 0.001 nM or lower, 0.005 nM or lower, 0.01 nM or lower, 0.05 nM or lower, about 0.1 nM or lower, about 0.5 nM or lower, about 1 nM or lower, about 5 nM or lower, about 10 nM or lower, about 50 nM or lower, about 100 nM or lower, about 500 nM or lower, or about 1 µM or lower.

In some embodiments, the Protein S antibodies provided herein are administered to a subject, wherein the antibody remains active in the subject for a period of time. In some embodiments, the period of time is antibody dose-dependent.

In some embodiments, the Protein S antibodies provided herein exhibit graded inhibition. In such embodiments, the graded inhibition can allow for increasing the dose of administration of the Protein S antibodies to a subject for added efficacy, or decreasing the dose to prevent excess thrombin generation, and/or potential thromboembolic complications, for example. Effectively, the dose can be adjusted to achieve the desired level of Protein S inhibition. Antibodies exhibiting graded inhibition show concentration-dependent inhibition in vitro, where increasing concentrations of antibody result in incremental increases in thrombin generation over a wide concentration range.

In some embodiments, the Protein S antibodies provided herein exhibit switch-like inhibition, wherein inhibition can be switched on or off. Antibodies exhibiting switch-like inhibition show abrupt concentration-dependent increases in thrombin generation in vitro, where upon reaching a concentration capable of promoting thrombin generation, achieves maximal thrombin generated within a narrow concentration range.

In some embodiments, the Protein S antibodies provided herein are antibody fragments. In some embodiments, the antibody fragments are antigen-binding fragments (Fab), variable fragments (Fv) containing VH and VL, single chain variable fragments (scFv) containing VH and VL linked together in one chain, or other antibody variable region fragments, such as Fab′, F(ab)2, F(ab′)2, dsFv diabody, Fc, and Fd polypeptide fragments. The antibody fragments may contain a Fc domain.

In some embodiments, the Protein S antibodies provided herein are monoclonal antibodies (mAbs). In some embodiments, the Protein S antibodies provided herein are human antibodies. In some embodiments, the Protein S antibodies provided herein are monoclonal human antibodies. In some embodiments, the Protein S antibodies provided herein are humanized antibodies. In some embodiments, the Protein S antibodies provided herein are monoclonal humanized antibodies. In some embodiments, the Protein S antibodies provided herein are chimeric antibodies. In some embodiments, the Protein S antibodies provided herein are monoclonal chimeric antibodies.

In some embodiments, the Protein S antibodies provided herein are full-length antibodies.

In some embodiments, the Protein S antibodies provided herein contain an Fc domain (either are full-length or are, for example, a single chain antibody linked to a Fc domain).

In some embodiments, the constant region (herein referred to also as a Fc domain, a Fc sequence or simply as a Fc) of a Protein S antibody is a human Fc domain. In some embodiments, the Fc domain of a full-length Protein S antibody is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the Fc domain of a full-length Protein S antibody is that of a rat. In some embodiments, the Fc domain of a full-length Protein S antibody is rat IgG1 or rat IgG2b. In some embodiments, the Fc domain of a full-length Protein S antibody is that of a non-human primate, e.g. it is a cynomolgus monkey Fc domain.

In some embodiments, the Protein S antibodies provided herein are chimeric and comprise a variable region from one species, and a constant region from another species, e.g. comprise a human variable region and a rat constant region. In some embodiments, the rat constant region is rat IgG1 or IgG2b. In some embodiments, the antibodies comprise a human variable region and a human constant region. In exemplary embodiments, the human constant region is human IgG1, or human IgG4.

In some embodiments, the Protein S antibody contains an Fc domain, and the Fc domain of the antibody is a human IgG1 Fc. Exemplary, but non-limiting, human IgG1 Fc domain sequences are provided as SEQ ID NOS: 138.

1 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 61 GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG 121 PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 181 STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 241 LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 301 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (SEQ ID NO: 138)

In some embodiments, the Protein S antibody contains an Fc domain, and the Fc domain of the antibody is a human IgG4 Fc. An exemplary human IgG4 heavy chain Fc domain sequence is provided as SEQ ID NO: 139.

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 139)

The EU numbering scheme is one of many available antibody numbering schemes based on the residue numbers assigned to a canonical antibody sequence. Accordingly, a skilled artisan would understand that reference to a particular residue using the EU numbering scheme may or may not be exactly the residue in one of the antibodies of the disclosure. For example, if a Protein S antibody of the disclosure comprises a V215A substitution in the Fc, wherein the position number of the amino acid residue is of the EU numbering scheme, the residue may not be the actual residue 215 in that particular Protein S antibody. It may be actual residue number 213, or 214, or 215, or 216. Accordingly a skilled artisan will understand how to correspond the recited residue using the EU numbering scheme, to the actual residue in a Protein S antibody of the disclosure. The EU numbering system for antibodies is known in the art and is described, for example, at imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html.

In some embodiments, the Fc domain of a Protein S antibody is an IgG1 or IgG4 human Fc domain, and Fc variants comprise at least one amino acid substitution at a position selected from the group consisting of: 215, 221, 222, 228, 234, 235, 236, 239, 240, 241, 243, 244, 245, 247, 250, 252, 254, 256, 262, 263, 264, 265, 266, 267, 268, 269, 270, 292, 296, 297, 298, 299, 300, 305, 313, 324, 325, 326, 327, 328, 329, 330, 332, 333, 334, 345, 396, 428, 430, 433, 434, and 440, wherein the position numbers of the amino acid residues are of the EU numbering scheme.

In some embodiments, the Fc domain of a Protein S antibody is a human IgG1, and substitutions are introduced to reduce effector function, including N297A, N297Q, N297G, L235E, L234A, and L235A, wherein the position numbers of the amino acid residues are of the EU numbering scheme. In some embodiments, the Fc domain of a full-length Protein S antibody is human IgG4, and substitutions are introduced to reduce effector function, including L235E, and F234A/L235A, wherein the position numbers of the amino acid residues are of the EU numbering scheme. In some embodiments, the Fc domain of a full-length Protein S antibody is human IgG2, and substitutions are introduced to reduce effector function, including H268Q/V309L/A330S/P331S and V234A/G237A/P238S/H268A/V309L/A330S/P331S, wherein the position numbers of the amino acid residues are of the EU numbering scheme.

In some embodiments, the Fc domain of a Protein S antibody is a human IgG1, and substitutions are introduced to increase effector function, including G236A/S239D,/I332E, K326W/E333S, S267E/H268F/S324T, and E345R/E430G/S440Y, wherein the position numbers of the amino acid residues are of the EU numbering scheme.

In some embodiments, the Fc domain of a Protein S antibody is an IgG4 human Fc domain, and the antibody is prone to the dynamic process of Fab-arm exchange. Accordingly, in some embodiments the IgG4 Fc domain comprises a S228P substitution, resulting in the reduction of this process, wherein the position number of the amino acid residues are of the EU numbering scheme.

In other embodiments, the Fc domain of a Protein S antibody is altered to increase its serum half-life. Such alterations include substitutions of a human IgG1 (e.g. SEQ ID NO: 138) such as T250Q/M428L, M252Y/S254T/T256E, M428L/N434S, S267E/L328F, N325S/L328F, and H433K/N434F, wherein the position number of the amino acid residues are of the EU numbering scheme.

In other embodiments, the Fc domain of a Protein S antibody is altered to increase its serum half-life. Such alterations include substitutions of a human IgG4 (e.g. SEQ ID NO: 139) such as T250Q/M428L, M252Y/S254T/T256E, M428L/N434S, S267E/L328F, N325S/L328F, and H433K/N434F, wherein the position number of the amino acid residues are of the EU numbering scheme.

In some embodiments, the Fc domain of a Protein S antibody is an IgG1 human Fc domain, and substitutions are introduced enhance effector function, including F243L/R292P/Y300L/V305I/P396L, S239D/I332E, S298A/E333A/K334A, L234Y/L235Q/G236W/S239M/H268D/D270E/S298A,D270E/K326D/A330M/K334E, wherein the position number of the amino acid residues are of the EU numbering scheme.

Exemplary Protein S Antibodies - Complementarity Determining Region (CDR) Sequences

Provided herein are exemplary CDR sequences of the Protein S antibodies disclosed herein.

Exemplary CDR sequences presented in Tables 1A-1C and 2A-2C below. As referred below, a light chain variable (VL) domain CDR1 region is referred to as CDR-L1; a VL CDR2 region is referred to as CDR-L2; a VL CDR3 region is referred to as CDR-L3; a heavy chain variable (VH) domain CDR1 region is referred to as CDR-H1; a VH CDR2 region is referred to as CDR-H2; and a VH CDR3 region is referred to as CDR-H3.

TABLE 1A Exemplary Protein S Antibody CDR-L1 Sequences QSVGSN (SEQ ID NO: 7) QSVRSN (SEQ ID NO: 8) QSIRSY (SEQ ID NO: 132)

TABLE 1B Exemplary Protein S Antibody CDR-L2 Sequences GAS (SEQ ID NO: 17) AAS (SEQ ID NO: 118)

TABLE 1C Exemplary Protein S Antibody CDR-L3 Sequences QQYNNWPLT (SEQ ID NO: 28) QQYNSWPFT (SEQ ID NO: 29) QQSYSTPRT (SEQ ID NO: 129)

TABLE 2A Exemplary Protein S Antibody CDR-H1 Sequences GYTFTSYD (SEQ ID NO: 40) RFSFSSYA (SEQ ID NO: 41) GFTLNSFW (SEQ ID NO: 133)

TABLE 2B Exemplary Protein S Antibody CDR-H2 Sequences VNPNSDNI (SEQ ID NO: 52) ISDSGSNT (SEQ ID NO: 53) IKEDGSEK (SEQ ID NO: 134)

TABLE 2C Exemplary Protein S Antibody CDR-H3 Sequences VRGAAAAWDAFAI (SEQ ID NO: 64) AKNYESGGYYTFFDY (SEQ ID NO: 65) ARETDILTAYYSDYYSYYGMDV (SEQ ID NO: 135)

In some embodiments, the Protein S antibodies provided herein include any one or more of the amino acid sequences of the CDR sequences provided in Tables 1A, 1B, 1C, 2A, 2B, and 2C.

In some embodiments, provided herein are Protein S antibodies, wherein the antibodies comprise:

-   (a) any one of the CDR-L1 amino acid sequences of Table 1A; -   (b) any one of the CDR-L2 amino acid sequences of Table 1B; -   (c) any one of the CDR-L3 amino acid sequences of Table 1C; -   (d) any one of the CDR-H1 amino acid sequences of Table 2A; -   (e) any one of the CDR-H2 amino acid sequences of Table 2B; -   (f) any one of the CDR-H3 amino acid sequences of Table 2C; and/or -   (g) any combination of the above.

In some embodiments, provided herein are Protein S antibodies, wherein the light chain variable domain of the antibodies comprises:

-   (a) a CDR-L1 comprising the amino acid sequence of any one of SEQ ID     NOs: 7, 8, and 132; -   (b) a CDR-L2 comprising the amino acid sequence of any one of SEQ ID     NOs: 17 and 118; and -   (c) a CDR-L3 comprising the amino acid sequence of any one of SEQ ID     NOs: 28, 29, and 129.

In some embodiments, provided herein are Protein S antibodies, wherein the heavy chain variable domain of the antibodies comprises:

-   (a) a CDR-H1 comprising the amino acid sequence of any one of SEQ ID     NOs: 40, 41, and 133; -   (b) a CDR-H2 comprising the amino acid sequence of any one of SEQ ID     NOs: 52, 53, and 134; and -   (c) a CDR-H3 comprising the amino acid sequence of any one of SEQ ID     NOs: 64, 65, and 135.

In some embodiments, provided herein are Protein S antibodies, wherein the light chain variable domain of the antibody comprises:

-   (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; -   (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17;     and -   (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

In some embodiments, provided herein are Protein S antibodies, wherein the light chain variable domain of the antibody comprises:

-   (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8; -   (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17;     and -   (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29.

In some embodiments, provided herein are Protein S antibodies, wherein the light chain variable domain of the antibody comprises:

-   (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 132; -   (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 118;     and -   (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 129.

In some embodiments, provided herein are Protein S antibodies, wherein the heavy chain variable domain of the antibodies comprises:

-   (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 40; -   (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52;     and -   (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 64.

In some embodiments, provided herein are Protein S antibodies, wherein the heavy chain variable domain of the antibodies comprises:

-   (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 41; -   (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 53;     and -   (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 65.

In some embodiments, provided herein are Protein S antibodies, wherein the heavy chain variable domain of the antibodies comprises:

-   (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 133; -   (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 134;     and -   (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 135.

In some embodiments, provided herein are Protein S antibodies, wherein the light chain variable domain of the antibodies comprises a CDR-L1 from Table 1A, a CDR-L2 from Table 1B, and a CDR-L3 from Table 1C, and wherein the heavy chain variable domain of the antibodies comprises:

-   (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 40; -   (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52;     and -   (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 64.

In some embodiments, provided herein are Protein S antibodies, wherein the light chain variable domain of the antibodies comprises a CDR-L1 from Table 1A, a CDR-L2 from Table 1B, and a CDR-L3 from Table 1C, and wherein the heavy chain variable domain of the antibodies comprises:

-   (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 41; -   (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 53;     and -   (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 65.

In some embodiments, provided herein are Protein S antibodies, wherein the light chain variable domain of the antibodies comprises a CDR-L1 from Table 1A, a CDR-L2 from Table 1B, and a CDR-L3 from Table 1C, and wherein the heavy chain variable domain of the antibodies comprises:

-   (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 133; -   (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 134;     and -   (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 135.

In some embodiments, provided herein are Protein S antibodies, wherein the heavy chain variable domain of the antibodies comprises a CDR-H1 from Table 2A, a CDR-H2 from Table 2B, and a CDR-H3 from Table 2C, and wherein the light chain variable domain of the antibodies comprises:

-   (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; -   (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17;     and -   (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

In some embodiments, provided herein are Protein S antibodies, wherein the heavy chain variable domain of the antibodies comprises a CDR-H1 from Table 2A, a CDR-H2 from Table 2B, and a CDR-H3 from Table 2C, and wherein the light chain variable domain of the antibodies comprises:

-   (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8; -   (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17;     and -   (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29.

In some embodiments, provided herein are Protein S antibodies, wherein the heavy chain variable domain of the antibodies comprises a CDR-H1 from Table 2A, a CDR-H2 from Table 2B, and a CDR-H3 from Table 2C, and wherein the light chain variable domain of the antibodies comprises:

-   (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 132; -   (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 118;     and -   (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 129.

In some embodiments, provided herein are Protein S antibodies, wherein the antibodies comprise the CDR amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 52, and SEQ ID NO: 64.

In some embodiments, provided herein are Protein S antibodies, wherein the antibodies comprise the heavy and light CDR amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 41, SEQ ID NO: 53, and SEQ ID NO: 65.

In some embodiments, provided herein are Protein S antibodies, wherein the antibodies comprise the CDR amino acid sequences of SEQ ID NO: 132, SEQ ID NO: 118, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135.

Exemplary Protein S Antibodies - Kappa and Lambda Light Chains

Provided herein are amino acid sequences for the kappa and lambda light chain constant regions of exemplary Protein S antibodies of the disclosure. The Protein S antibodies provided herein may have a kappa light chain constant region or a lambda light chain constant region. The sequences of the kappa and lambda light chain constant regions are provided in Table 3.

TABLE 3 Kappa light chain sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 214) Lambda light chain sequence GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHR SYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 215)

In exemplary embodiments, the Protein S antibody having the light chain sequence of SEQ ID NO: 214 comprises CDRs having the amino acid sequences as set forth in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 52, and SEQ ID NO: 64.

In exemplary embodiments, the Protein S antibody having the light chain sequence of SEQ ID NO: 215 comprises CDRs having the amino acid sequences as set forth in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 52, and SEQ ID NO: 64.

In exemplary embodiments, the Protein S antibody having the light chain sequence of SEQ ID NO: 214 comprises CDRs having the amino acid sequences as set forth in SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 41, SEQ ID NO: 53, and SEQ ID NO: 65.

In exemplary embodiments, the Protein S antibody having the light chain sequence of SEQ ID NO: 215 comprises CDRs having the amino acid sequences as set forth in SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 41, SEQ ID NO: 53, and SEQ ID NO: 65.

In exemplary embodiments, the Protein S antibody having the light chain sequence of SEQ ID NO: 214 comprises CDRs having the amino acid sequences as set forth in SEQ ID NO: 132, SEQ ID NO: 118, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135.

In exemplary embodiments, the Protein S antibody having the light chain sequence of SEQ ID NO: 215 comprises CDRs having the amino acid sequences as set forth in SEQ ID NO: 132, SEQ ID NO: 118, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135.

Exemplary Protein S Antibodies - Variable Region Sequences

Provided herein are amino acid sequences for the variable domains of exemplary Protein S antibodies of the disclosure. The exemplary variable light chain amino acid sequences and exemplary variable heavy chain amino acid sequences are presented in Tables 4A and 4B below. Table 4C presents exemplary combinations of variable heavy and variable light chains.

Accordingly, in some embodiments, the Protein S antibodies of the disclosure comprise the variable chain amino acid sequence of any one of the combinations provided in Table 4C. In some embodiments, provided herein are Protein S antibodies, wherein the antibodies comprise a variable light chain comprising the amino acid sequence selected from SEQ ID NO: 83, 85, and 154. In some embodiments, provided herein are Protein S antibodies, wherein the antibodies comprise a variable heavy chain comprising the amino acid sequence selected from SEQ ID NO: 84, 86, and155.

TABLE 4A Exemplary Variable Light Chain Amino Acid Sequences of Protein S Monoclonal Antibodies EIVMTQSPATLSVSPGERATLSCRASQSVGSNLAWYQQKPGQAPRLLIYGAST RATGGPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPLTFGGGTKVEI K (SEQ ID NO: 83) EIVMTQSPATLSVFPGERATLSCRASQSVRSNLAWHQQKLGQAPRLLLYGAS TRATGIPARFSGSGSGTEFTLAISSLQSEDFAVYYCQQYNSWPFTFGGGTKVGI K (SEQ ID NO: 85) DIQMTQSPSSLSASVRDRVTINCRASQSIRSYLNWYQQKPGKAPKLLIYAASSL QGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPRTFGQGTKVEIK (SEQ ID NO: 154)

TABLE 4B Exemplary Variable Heavy Chain Amino Acid Sequences of Protein S Monoclonal Antibodies QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGW VNPNSDNIGYAQKFQGRVTMTRNTSISTAYMELSSLRSEDSAVYYCVRGAAA AWDAFAIWGQGTMVTVSS (SEQ ID NO: 84) EVQLLESGGGLVQPGGSLRLSCAASRFSFSSYAMSWVRQAPGKGLEWVSAIS DSGSNTYYADSVKGLFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNYESG GYYTFFDYWGQGTLVTVST (SEQ ID NO: 86) EVQVVESGGGLVQPGGSLRLSCAASGFTLNSFWMSWVRQAPGKGLEWVANI KEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNGLRAEDTALYYCARETDIL TAYYSDYYSYYGMDVWGQGTTVTVSA (SEQ ID NO: 155)

TABLE 4C Exemplary Variable Light Chain and Variable Heavy Chain Amino Acid Sequences of Protein S Monoclonal Antibodies Combination Number Variable Light Chain Amino Acid Sequence Variable Heavy Chain Amino Acid Sequence Combination 8 SEQ ID NO: 83 SEQ ID NO: 84 Combination 9 SEQ ID NO: 85 SEQ ID NO: 86 Combination 16 SEQ ID NO: 154 SEQ ID NO: 155

In some embodiments, the heavy and light chain variable domains of the Protein S antibodies provided herein include the amino acid sequence of any one of the numbered combinations presented in Table 4C.

In some embodiments, provided herein are Protein S antibodies, wherein the light chain variable domain of the antibodies comprise the amino acid sequence of SEQ ID NO: 83 or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, provided herein are Protein S antibodies, wherein the heavy chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 84, or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, provided herein are Protein S antibodies, wherein the light chain variable domain of the antibodies comprise the amino acid sequence of SEQ ID NO: 83 or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 84, or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In such embodiments, the antibodies may comprise the CDR amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 52, and SEQ ID NO: 64.

In some embodiments, provided herein are Protein S antibodies, wherein the light chain variable domain of the antibodies comprise the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, provided herein are Protein S antibodies, wherein the heavy chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 86, or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, provided herein are Protein S antibodies, wherein the light chain variable domain of the antibodies comprise the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 86, or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In such embodiments, the antibodies may comprise the CDR amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 41, SEQ ID NO: 53, and SEQ ID NO: 65.

In some embodiments, provided herein are Protein S antibodies, wherein the light chain variable domain of the antibodies comprise the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, provided herein are Protein S antibodies, wherein the heavy chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 155, or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, provided herein are Protein S antibodies, wherein the light chain variable domain of the antibodies comprise the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 155, or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In such embodiments, the antibodies may comprise the CDR amino acid sequences of SEQ ID NO: 132, SEQ ID NO: 118, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135.

In some embodiments, the Protein S antibodies lead to a decrease in levels of circulating TFPI. Exemplary antibodies that lead to a decrease in levels of circulating TFPI (1) comprise the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 83, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 84; (2) comprise the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 85, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 86; (3) comprise the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 154, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 155; (4) comprise the light and heavy CDR amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 52, and SEQ ID NO: 64; (5) comprise the light and heavy CDR amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 41, SEQ ID NO: 53, and SEQ ID NO: 65; and (6) comprise the light and heavy CDR amino acid sequences of SEQ ID NO: 132, SEQ ID NO: 118, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135.

In some embodiments, the Protein S antibodies increase fibrin deposition. Exemplary antibodies that increase fibrin deposition (1) comprise the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 83, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 84; (2) comprise the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 85, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 86; (3) comprise the light and heavy CDR amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 52, and SEQ ID NO: 64; and (4) comprise the light and heavy CDR amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 41, SEQ ID NO: 53, and SEQ ID NO: 65.

In some embodiments, the Protein S antibodies provided promote thrombin generation. Exemplary antibodies that promote thrombin generation (1) comprise the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 83, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 84; (2) comprise the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 85, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 86; (3) comprise the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 154, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 155; (4) comprise the light and heavy CDR amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 52, and SEQ ID NO: 64; (5) comprise the light and heavy CDR amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 41, SEQ ID NO: 53, and SEQ ID NO: 65; and (6) comprise the light and heavy CDR amino acid sequences of SEQ ID NO: 132, SEQ ID NO: 118, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135.

In some embodiments, the Protein S antibodies provided herein prevent the interaction between TFPI and Protein S. Exemplary antibodies that prevent TFPI and Protein S interaction (1) comprise the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 83, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 84; (2) comprise the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 85, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 86; (3) comprise the light and heavy CDR amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 52, and SEQ ID NO: 64; and (4) comprise the light and heavy CDR amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 41, SEQ ID NO: 53, and SEQ ID NO: 65.

As noted above, Protein S comprises four domains: the γ-carboxγ-glutamic acid domain (Gla-domain), the thrombin-sensitive region (TSR), the epidermal growth factor-like domain (EGF domain), and the sex hormone binding globulin-like domain (SHBG domain). FIG. 1 depicts the schematic diagram of Protein S showing these modular domains of Protein S. The TSR is within the heavy chain of Protein S. The heavy chain of Protein S represents amino acids 42-296 of Protein S, the TSR represents amino acids 88-116, the signal peptide represents amino acids 1-24 and the propeptide represents amino acids 25-41.

In some embodiments, the Protein S antibodies provided herein do not bind the Protein S heavy chain. Exemplary antibodies that do not bind to the Protein S heavy chain (1) comprise the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 83, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 84; (2) comprise the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 85, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 86; (3) comprise the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 154, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 155; (4) comprise the light and heavy CDR amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 52, and SEQ ID NO: 64; (5) comprise the light and heavy CDR amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 41, SEQ ID NO: 53, and SEQ ID NO: 65; and (6) comprise the light and heavy CDR amino acid sequences of SEQ ID NO: 132, SEQ ID NO: 118, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135.

The Protein S antibodies provided herein bind Protein S at a region other than the TSR. In some embodiments, the Protein S antibodies provided herein bind to the heavy chain of Protein S, but do not bind the TSR region of the heavy chain. In some embodiments, the Protein S antibodies provided are inhibitors of TFPI, and do not bind to the TSR of Protein S.

In some embodiments, the Protein S antibodies provided herein do not bind to the TSR of Protein S, and cause an inhibition of the activity of TFPI, disrupt the binding of Protein S-TFPI, prevent the formation of a Protein S-TFPI complex, and/or lead to a decrease in circulating levels of TFPI.

In some embodiments, the Protein S antibodies provided herein bind to a conformational epitope, i.e., an epitope that is non-linear.

As discussed above, Protein S can be found in a free form, or in a complexed form. Protein S can form a complex with at least TFPI, and with C4b-binding protein (C4BP).

In some embodiments, the Protein S antibodies provided herein are capable of binding to Protein S in a free form and a complexed form, e.g. complexed with TFPI or C4BP. In some embodiments, the Protein S antibodies provided herein are capable of binding to Protein S that is either in free or complexed form.

In some embodiments, the Protein S antibodies provided herein bind to a complexed Protein S, wherein the Protein S is bound to TFPI. In some embodiments, the Protein S antibodies provided herein bind to a complexed Protein S, wherein the Protein S is bound to C4BP. In some embodiments, the Protein S antibodies provided herein prevent the formation of a complex between Protein S and TFPI.

In some embodiments, the Protein S antibodies provided herein bind Protein S, and the binding affinity of the antibodies to Protein S is calcium-independent. Exemplary antibodies of the disclosure that show binding to Protein S that is calcium-independent comprise the CDR amino acid sequences of (1) SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 52, and SEQ ID NO: 64; (2) SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 41, SEQ ID NO: 53, and SEQ ID NO: 65; and (3) SEQ ID NO: 132, SEQ ID NO: 118, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135.

Graded inhibition can be observed with at least antibodies that (1) comprise the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 83, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 84; (2) comprise the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 85, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 86; (3) comprise the light and heavy CDR amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 52, and SEQ ID NO: 64; and (4) comprise the light and heavy CDR amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 41, SEQ ID NO: 53, and SEQ ID NO: 65.

B. Generation of Protein S Antibodies

Production of the Protein S antibodies provided herein may be by use of any method known to those of ordinary skill in the art. In some embodiments, the antibodies are produced by hybridomas. In some embodiments, the antibodies are encoded by a nucleic acid and are expressed, purified, and isolated.

Accordingly, provided herein are nucleic acids encoding any of the antibodies disclosed herein, vectors comprising any of the nucleic acids encoding such antibodies, and host cells comprising such vectors. Also provided herein are exemplary nucleic acid sequences encoding for the variable heavy chains and variable light chains of the Protein S antibodies disclosed herein.

Tables 5A and 5B provide exemplary variable light chain nucleic acid sequences and exemplary variable heavy chain nucleic acid sequences. Exemplary combinations of nucleic acid sequences encoding for the variable heavy and light chain domains of the Protein S antibodies disclosed herein are presented in Table 5C. The exemplary amino acid sequences of Tables 4A-4C correspond to the nucleic acid sequences of Tables 5A-5C. The exemplary combinations of Table 5C correspond to the numbered combinations presented in Table 4C.

The person of ordinary skill in the art will appreciate that, because of redundancy in the triplet code, multiple nucleic acids may encode the same amino acid sequence. Thus, nucleic acid sequences that are not identical to those set forth in the tables below may still encode the Protein S antibodies of the disclosure.

TABLE 5A Exemplary Variable Light Chain Nucleic Acid Sequences of Anti-Protein S Monoclonal Antibodies GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGAGA AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGCAACTTAG CCTGGTACCAGCAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGT GCGTCCACCAGGGCCACTGGTGGCCCAGCCAGGTTCAGTGGCAGTGGGTC TGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGC AGTTTACTACTGTCAGCAGTATAATAACTGGCCTCTCACTTTCGGCGGAGG GACCAAGGTGGAGATCAAA (SEQ ID NO: 107) GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTTTCCAGGGGA AAGAGCCACCCTCTCTTGTAGGGCCAGTCAGAGTGTTAGAAGCAACTTAG CCTGGCACCAGCAGAAACTTGGCCAGGCTCCCAGGCTCCTCCTCTATGGA GCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTC TGGGACAGAGTTCACTCTCGCCATCAGCAGCCTGCAGTCTGAAGATTTTGC

TABLE 5A Exemplary Variable Light Chain Nucleic Acid Sequences of Anti-Protein S Monoclonal Antibodies AGTTTATTACTGTCAGCAGTATAATAGCTGGCCTTTCACTTTCGGCGGAGG GACCAAGGTGGGGATCAAA (SEQ ID NO: 109) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAAGAGAC AGAGTCACCATCAATTGCCGGGCAAGTCAGAGCATTAGGAGCTATTTAAA TTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTG CATCCAGTTTGCAAGGTGGGGTCCCATCACGGTTCAGTGGCAGTGGATCT GGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCA ACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGACGTTCGGCCAAGG GACCAAGGTGGAAATCAAA (SEQ ID NO: 168)

TABLE 5B Exemplary Variable Heavy Chain Nucleic Acid Sequences of Anti-Protein S Monoclonal Antibodies CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTC AGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGTTATGATAT CAACTGGGTGCGACAGGCCACTGGACAAGGCCTTGAGTGGATGGGATGGG TGAACCCTAACAGTGATAACATAGGCTATGCACAGAAGTTCCAGGGCAGA GTCACCATGACCAGGAACACCTCCATAAGCACAGCCTACATGGAACTGAG CAGCCTGAGATCTGAGGACTCGGCCGTGTATTATTGTGTGAGAGGAGCAG CAGCAGCTTGGGATGCTTTTGCTATCTGGGGCCAAGGGACAATGGTCACC GTCTCTTCA (SEQ ID NO: 108) GAGGTGCAGTTGCTGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGGGTC CCTGAGACTCTCCTGTGCAGCCTCTAGATTCAGCTTTAGCAGCTACGCCAT GAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGTCTCAGCCA TTAGTGACAGTGGTAGTAACACATACTACGCAGACTCCGTGAAGGGCCTA TTCACCATCTCCAGAGACAATTCCAAGAACACGTTGTATCTGCAAATGAA CAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAACTATG AGTCGGGGGGTTATTATACCTTCTTTGACTACTGGGGCCAGGGAACCCTGG TCACCGTCTCCACA (SEQ ID NO: 110) GAGGTGCAGGTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTC CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCCTTAATAGTTTTTGGAT GAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAAC ATAAAGGAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCC GATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATG AACGGCCTGAGAGCCGAGGACACGGCTCTGTATTACTGTGCGAGAGAAAC CGATATTTTGACTGCTTATTATTCGGATTACTACTCCTATTACGGTATGGAC GTCTGGGGCCAAGGGACCACGGTCACCGTCTCCGCA (SEQ ID NO: 169)

TABLE 5C Exemplary Variable Light Chain and Variable Heavy Chain Nucleic Acid Sequences of Anti-Protein S Monoclonal Antibodies Combination Number Variable Light Chain Nucleic Acid Sequence Variable Heavy Chain Nucleic Acid Sequence Combination 8 SEQ ID NO: 107 SEQ ID NO: 108 Combination 9 SEQ ID NO: 109 SEQID NO: 110 Combination 16 SEQ ID NO: 168 SEQ ID NO: 169

In some embodiments, provided herein are nucleic acids encoding any of the Protein S antibodies disclosed herein. In some embodiments, provided herein are nucleic acids comprising any one or more of the nucleic acid sequences of Tables 5A-5B. In some embodiments, the heavy chain and light chain variable domains of the Protein S antibodies disclosed herein are encoded by a nucleic acid comprising any one or more of the nucleic acid sequences of Tables 5A-5B.

In some embodiments, the variable domain of the Protein S antibodies of the disclosure are encoded by a nucleic acid, wherein the light chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 107 or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, the variable domain of the Protein S antibodies of the disclosure are encoded by a nucleic acid, wherein the heavy chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 108, or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, the variable domain of the Protein S antibodies of the disclosure are encoded by a nucleic acid, wherein the light chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 107 or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 108, or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, the variable domain of the Protein S antibodies of the disclosure are encoded by a nucleic acid, wherein the light chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 109 or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, the variable domain of the Protein S antibodies of the disclosure are encoded by a nucleic acid, wherein the heavy chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 110, or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, the variable domain of the Protein S antibodies of the disclosure are encoded by a nucleic acid, wherein the light chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 109 or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 110, or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, the variable domain of the Protein S antibodies of the disclosure are encoded by a nucleic acid, wherein the light chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 168 or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, the variable domain of the Protein S antibodies of the disclosure are encoded by a nucleic acid, wherein the heavy chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 169, or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, the variable domain of the Protein S antibodies of the disclosure are encoded by a nucleic acid, wherein the light chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 168 or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 169, or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

The disclosure also provides vectors comprising any nucleic acid of the disclosure. In some embodiments, the nucleic acid of the vector comprises any one or more of the nucleic acid sequences selected from Tables 5A-5B. In some embodiments, the vector is an expression vector or an expression construct. In some embodiments, the vector is a mammalian vector. In some embodiments, the vector is a viral vector.

In some embodiments, the Protein S antibodies provided herein are produced by culturing a cell under suitable conditions for leading to the expression of the Protein S antibody, wherein the cell comprises a vector.

II. Uses of Protein S Antibodies A. Therapeutic Protein S Antibodies

Provided herein are antibodies that recognize and selectively and/or specifically bind to Protein S, including soluble Protein S (free Protein S), and Protein S that is part of a complex (e.g. with TFPI, or C4BP). The antibodies disclosed herein may be used for therapeutics in a subject. In some embodiments, the subject is a mammalian subject. In some embodiments, the mammalian subject is a human subject. In some embodiments, the mammalian subject is a non-human primate, e.g. a cynomolgus monkey.

In some embodiments, the Protein S antibodies provided herein are useful for treating a condition in a subject, wherein the condition is associated with the coagulation cascade. In some embodiments, the Protein S antibodies provided herein are useful for reducing an ability of Protein S to act as a cofactor within the coagulation cascade for the treatment of a condition in a subject.

In some embodiments, the Protein S antibodies provided herein are useful for reducing an ability of Protein S to act as a cofactor for TFPI for the treatment of a bleeding disorder or other diseases, e.g., a platelet disorder, by promoting thrombin generation.

In some embodiments, provided herein is a method of promoting the coagulation of blood, the method comprising contacting any one of the Protein S antibodies disclosed herein with Protein S. In some embodiments, the contacting takes place in plasma. In some embodiments, the method is in vitro. In some embodiments, the method is in vivo. In some embodiments, the method is in vivo, and the method further comprises administering any one of the Protein S antibodies disclosed herein to a subject in need thereof.

In some embodiments, provided herein is a method of promoting the coagulation of blood, decreasing TFPI levels, disrupting the complexation of TFPI and Protein S, and/or preventing the formation of a Protein S-TFPI complex, the method comprising contacting any one of the Protein S antibodies disclosed herein with a blood sample. In some embodiments, the result is marked by an increase in thrombin generation. In some embodiments, the result is marked by an increase in fibrin generation.

In some embodiments provided herein are antibodies that bind Protein S, wherein the binding of the antibody to Protein S decreases circulating TFPI levels.

In some embodiments provided herein are antibodies that bind Protein S, wherein the binding of the antibody to Protein S decreases or disrupts a Protein S-TFPI complex, or prevents the formation of a Protein S-TFPI complex.

In some embodiments, the effects of the antibody is marked by an increase in D-dimer.

In some embodiments, provided herein is a method of promoting the coagulation of blood in a subject in need thereof, the method comprising administering to the subject any one of the Protein S antibodies disclosed herein, or any one of the pharmaceutical compositions disclosed herein. In some embodiments, the antibody remains active in the subject for a period of time, wherein the period of time is antibody dose-dependent. In some embodiments, the period of time is about 50 hours to about 170 hours. In some embodiments, the antibody exhibits switch-like inhibition. In some embodiments, the antibody exhibits graded inhibition.

In some embodiments, provided herein is a method of decreasing circulating levels of TFPI in a subject in need thereof, the method comprising administering to the subject any one of the Protein S antibodies disclosed herein, or any one of the pharmaceutical compositions disclosed herein. In some embodiments, the subject suffers from a disease or condition selected from the group consisting of bleeding disorders, and platelet disorders. In some embodiments, the subject suffers from trauma and/or bleeding resulting from a surgery or a medical procedure. For example, the medical procedure may be a procedure in which bleeding may occur, but not necessarily so. In some embodiments, the medical procedure is a dental procedure.

In some embodiments, provided herein is a method of promoting generation of thrombin in a subject in need thereof, the method comprising administering to the subject any one of the Protein S antibodies disclosed herein, or any one of the pharmaceutical compositions disclosed herein. In some embodiments, the subject suffers from a disease or condition selected from the group consisting of bleeding disorders, and platelet disorders. In some embodiments, the subject suffers from trauma and/or bleeding resulting from a surgery or a medical procedure. For example, the medical procedure may be a procedure in which bleeding may occur, but not necessarily so. In some embodiments, the medical procedure is a dental procedure.

In some embodiments, provided herein is a method of treating a condition in a subject in need thereof, wherein the disease is selected from the group consisting of bleeding disorders, and platelet disorders, and the method comprises administering to the subject any one of the Protein S antibodies disclosed herein, or any one of the pharmaceutical compositions disclosed herein. In some embodiments, the condition is a bleeding disorder. In some embodiments, the bleeding disorder is selected from the group consisting of hemophilia A, hemophilia B, von Willebrand disease (vWD, which may be a subtype selected from: vWD Type 1, vWD Type 2A, vWD Type 2B, vWD Type 2N, vWD Type 2M, vWD Type 3, and acquired vWD), menorrhagia including menorrhagia due to a congenital or acquired factor deficiency, Factor I deficiency, Factor II deficiency, Factor V deficiency, Factor VII deficiency, Factor X deficiency, Factor XI deficiency (hemophilia C), Factor VIII deficiency (hemophilia A), Factor IX deficiency (hemophilia B), trauma, and hereditary hemorrhagic telangiectasia. In some embodiments, the bleeding is associated with surgery, e.g. in a subject with a type of hemophilia. In some embodiments, the bleeding is associated with a medical procedure, e.g., a dental procedure. In some embodiments, the bleeding disorder is vWD, and the subject also suffers from menorrhagia. In some embodiments, the bleeding disorder is vWD, and the subject is undergoing a prophylactic treatment. In some embodiments, the subject suffers from menorrhagia associated with any one or more bleeding disorders and/or platelet disorders. In some embodiments, the subject is a hemophilia carrier. In some embodiments, the subject is a hemophilia carrier, and suffers from menorrhagia. In some embodiments, the condition is a platelet disorder. In some embodiments, the platelet disorder includes but is not limited to Bernard-Soulier syndrome, Glanzmann’s thrombasthenia, and platelet storage pool deficiencies. In some embodiments, the platelet disorder is a platelet storage pool deficiency. In some embodiments, the platelet storage pool deficiency includes but is not limited to: Gray platelet syndrome, Quebec platelet disorder, and MYH9-related thrombocytopenia (MYH9RD). In some embodiments, the subject has a bleeding disorder, and has inhibitors. In some embodiments, the bleeding disorder is hemophilia A or hemophilia B, wherein the subject has inhibitors. In some embodiments, the bleeding disorder is vWD. For example, the inhibitors may be developed in the subject as a response to factor replacement therapy.

In some embodiments, a subject in need thereof may be treated with any of the Protein S antibodies provided herein, wherein the treatment is a routine prophylaxis to prevent or reduce the frequency of bleeding episodes. In some embodiments, a subject in need thereof may be treated with any of the Protein S antibodies provided herein, wherein the treatment is an on-demand treatment used for the control of bleeding episodes. In some embodiments, a subject in need thereof may be treated with any of the Protein S antibodies provided herein, wherein the treatment is a perioperative management of bleeding. For example, a perioperative management treatment may be used for treating a subject prior to, during, and/or after surgery or other medical procedure, or prior to, during, and/or after trauma.

In some embodiments, the treatment with any of the Protein S antibodies provided herein is provided as a chronic therapy, with dosing occurring continuously over time. In some embodiments, the treatment with any of the Protein S antibodies provided herein is provided as an intermittent therapy, with dosing occurring at irregular intervals. As an example, such an intermittent therapy can be used for a subject having menorrhagia. In some embodiments, the treatment with any of the Protein S antibodies provided herein is provided as an acute therapy, with dosing occurring for a short finite period of time. For example, the acute therapy may be administered for spontaneous bleeding episodes, or in conjunction with a surgery or other medical procedure, or after experiencing a trauma.

In some embodiments, the method of treatment of a subject may be a combination one of the above, e.g., the method of treatment may be prophylactic, and on-demand. In some embodiments, a prophylactic method of treatment may be a chronic therapy. In some embodiments, a prophylactic method of treatment may be an acute therapy. In some embodiments, a prophylactic method of treatment may be an intermittent therapy. In some embodiments, an on-demand treatment may be an acute therapy. In some embodiments, an on-demand treatment may be an intermittent treatment.

In some embodiments, treatment of a subject in need thereof comprises administering to the subject any of the Protein S antibodies provided herein, wherein the Protein S antibodies provided herein are Fab fragments. Without being bound to any theory, in some embodiments a shorter half-life of a Fab fragment, in relation to a full-length antibody with the same VH/VL may be beneficial for an acute treatment or on-demand. In some embodiments, the Fab fragment Protein S antibodies are administered to a subject in need thereof to reduce risk of bleeding.

B. Combination Therapies

The administration of any one of the therapeutic Protein S antibodies provided herein may be a monotherapy, or may be in combination with any other known drugs or treatments for diseases or conditions. In some embodiments, the other known drugs or treatments are useful for treating disorders, diseases, or conditions associated with reduced or impaired clotting. In some embodiments, the disorder, condition is a bleeding disorder. In some embodiments, the disorder, disease, or condition is a bleeding disorder or a platelet disorder.

In some embodiments, the administration of any of the therapeutic Protein S antibodies provided herein may be with a factor replacement therapy. In some embodiments, the administration of any of the therapeutic Protein S antibodies provided herein may be with the administration of a recombinant Factor VIIa.

C. Administration of Therapeutic Protein S Antibodies

The in vivo administration of the therapeutic Protein S antibodies described herein may be carried out intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, intrathecally, intraventricularly, intranasally, transmucosally, through implantation, or through inhalation. Administration of the therapeutic Protein S antibodies may be performed with any suitable excipients, carriers, or other agents to provide suitable or improved tolerance, transfer, delivery, and the like.

In some embodiments, the in vivo administration of any of the therapeutic Protein S antibodies provided herein may be an intravenous administration. In some embodiments, the intravenous administration may be provided as a prophylactic treatment. In some embodiments, the prophylactic treatment may be a routine prophylaxis. In some embodiments, the routine prophylaxis may have a regular dosing schedule. In some exemplary embodiments, the regular dosing schedule may be once weekly, twice weekly, once monthly, twice monthly, or three times monthly. In some embodiments, the intravenous administration may be provided as an on-demand treatment. In some embodiments, the intravenous administration may be provided as a chronic therapy. In some embodiments, the intravenous administration may be provided as an intermittent therapy. In some embodiments, the intravenous administration may be provided as an acute therapy. In some embodiments, an intermittent therapy may have a regular dosing schedule for the duration of the intermittent therapy. In some embodiments, an acute therapy may have a regular dosing schedule for the duration of the acute therapy. For example, administration of any of the therapeutic Protein S antibodies provided herein for an acute therapy by intravenous administration may occur on a regular dosing schedule for a predetermined duration of days, e.g., 7 days, 14 days, or more.

In some embodiments, the in vivo administration of any of the therapeutic Protein S antibodies provided herein may be a subcutaneous administration. In some embodiments, the subcutaneous administration may be provided as a prophylactic treatment. In some embodiments, the prophylactic treatment may be a routine prophylaxis. In some embodiments, the routine prophylaxis may have a regular dosing schedule. In some exemplary embodiments, the regular dosing schedule may be once weekly, twice weekly, once monthly, twice monthly, or three times monthly. In some embodiments, the subcutaneous administration may be provided as an on-demand treatment. In some embodiments, the subcutaneous administration may be provided as a chronic therapy. In some embodiments, the subcutaneous administration may be provided as an intermittent therapy. In some embodiments, the subcutaneous administration may be provided as an acute therapy. In some embodiments, an intermittent therapy may have a regular dosing schedule for the duration of the intermittent therapy. In some embodiments, an acute therapy may have a regular dosing schedule for the duration of the acute therapy. For example, administration of any of the therapeutic Protein S antibodies provided herein for an acute therapy by subcutaneous administration may occur on a regular dosing schedule for a predetermined duration of days, e.g., 7 days, 14 days, or more.

In some embodiments, the Protein S antibodies provided herein may be provided in a small volume amenable for injection, such as for subcutaneous administration. In some embodiments, the Protein S antibodies provided herein may be provided in a small volume amenable for injection by use of a pen-like auto-injector device. In some embodiments, the device is a syringe, for example a pre-filled syringe.

In some embodiments provided herein are single-dose vials useful for either subcutaneous or intravenous administration.

Accordingly, a therapeutically effective amount of the Protein S antibodies provided herein may be provided in a small volume for subcutaneous administration to a subject in need thereof. In some embodiments, the Protein S antibodies provided herein may be provided in a large volume amenable for administration by a subcutaneous infusion device, for subcutaneous infusion to a subject in need thereof.

D. Pharmaceutical Compositions

The disclosure also provides pharmaceutical compositions comprising any one of the Protein S antibodies disclosed herein, and optionally a pharmaceutical acceptable excipient or carrier. In some embodiments, the pharmaceutical composition is sterile. The pharmaceutical compositions may be formulated to be compatible with their intended routes of administration. In some embodiments, the pharmaceutical compositions of the disclosure are suitable for administration to a human subject.

E. Diagnostic Antibodies

The antibodies provided herein may also be used for diagnostic purposes. For example, diagnostic antibodies could be used for detecting protein S deficiencies, or for detecting protein S levels in plasma prior to dosing (e.g. as a companion diagnostic).

Accordingly, in some embodiments, a Protein S antibody of the disclosure is conjugated to a label, for example a detectable label, a spin label, a colorimetric label, a radioactive label, an enzymatic label, a fluorescent label, or a magnetic label. The label may be detectable by spectroscopic, photochemical, biochemical, immunochemical, fluorescent, electrical, optical or chemical methods. Useful labels include, but are not limited to, magnetic beads (e.g. DYNABEADS®), fluorescent dyes (e.g., fluorescein isothiocyanate, red, rhodamine, and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., LacZ, CAT, horseradish peroxidase, alkaline phosphatase and others, commonly used as detectable enzymes, either as marker gene products or in an ELISA), biotin, avidin, or streptavidin and colorimetric labels such as colloidal gold colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads, and nanoparticles. In some embodiments, provided herein are substrates to which one or more Protein S antibodies of the disclosure is attached.

Detection may be carried out on any biological sample obtained from a subject. Biological samples include, but are not limited to whole blood, plasma, serum, saliva, urine, feces, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural effusion, tissue, cells, a biopsy, interstitial fluid, lymphatic fluid, or fractions thereof derived from a subject. In some embodiments, the biological sample comprises cells and the cells are in culture, in a suspension, on a slide, in intact tissue, or in preparation ready for a FACs analysis.

III. Kits and Articles of Manufacture

The disclosure also provides a kit or article of manufacture comprising any one of the antibodies disclosed herein, or any pharmaceutical composition disclosed herein. In some embodiments, the kits may further include instructional materials for carrying out any of the methods disclosed herein. In some embodiments, the kits may further include sterile containers or vials for holding the antibodies and/or pharmaceutical compositions disclosed herein. In some embodiments, the kits may further include sterile delivery devices for administering the antibodies and/or pharmaceutical compositions disclosed herein. In some embodiments, an article of manufacture comprises any pharmaceutical composition of the disclosure.

IV. Exemplary Enumerated Embodiments

Exemplary enumerated embodiments of the disclosure are as follows.

Exemplary enumerated embodiments of the disclosure are as follows.

Embodiment I-1. An antibody that binds Protein S, wherein the binding of the antibody to Protein S decreases circulating tissue factor pathway inhibitor (TFPI) levels.

Embodiment I-2. An antibody that binds Protein S, wherein the binding of the antibody to Protein S disrupts a Protein S-TFPI complex, or prevents the formation of a Protein S-TFPI complex.

Embodiment I-3. The antibody of any one of embodiments I-1 to I-2, wherein the antibody is an inhibitor for the cofactor activity of Protein S for TFPI.

Embodiment I-4. The antibody of any one of embodiments I-1 to I-3, wherein the binding of the antibody to Protein S induces at least a 10% decrease in circulating TFPI levels.

Embodiment I-5. The antibody of any one of embodiments I-1 to I-4, wherein the binding of the antibody to Protein S disrupts the Protein S-TFPI complex.

Embodiment I-6. The antibody of any one of embodiments I-1 to I-5, wherein the binding of the antibody to Protein S prevents the formation of the Protein S-TFPI complex.

Embodiment I-7. The antibody of any one of embodiments I-1 to I-6, wherein the antibody binds to the C-terminus of Protein S.

Embodiment I-8. The antibody of any one of embodiments I-1 to I-7, wherein the antibody binds to an sex hormone binding globulin-like domain (SHBG-like domain) of Protein S.

Embodiment I-9. The antibody of any one of embodiments I-1 to I-8, wherein the binding of the antibody to Protein S promotes generation of a marker associated with coagulation activity.

Embodiment I-10. The antibody of any one of embodiments I-1 to I-9, wherein the binding of the antibody to Protein S promotes thrombin generation.

Embodiment I-11. The antibody of any one of embodiments I-1 to I-10, wherein the binding of the antibody to Protein S promotes D-dimer levels.

Embodiment I-12. The antibody of any one of embodiments I-1 to I-11, wherein the binding of the antibody to Protein S promotes fibrin generation.

Embodiment I-13. The antibody of any one of embodiments I-1 to I-12, wherein the activity of the antibody is dose-dependent.

Embodiment I-14. The antibody of any one of embodiments I-1 to I-13, wherein activity of the antibody is measured in vitro.

Embodiment I-15. The antibody of any one of embodiments I-1 to I-13, wherein activity of the antibody is measured in vivo.

Embodiment I-16. The antibody of any one of embodiments I-1 to I-15, wherein the antibody binds to free Protein S.

Embodiment I-17. The antibody of any one of embodiments I-1 to I-15, wherein the antibody binds to complexed Protein S.

Embodiment I-18. The antibody of embodiment I-17, wherein the Protein S is complexed with C4BP.

Embodiment I-19. The antibody of embodiment I-17, wherein the Protein S is complexed with TFPI.

Embodiment I-20. The antibody of any one of embodiments I-1 to I-19, wherein the antibody is a monoclonal antibody.

Embodiment I-21. The antibody of any one of embodiments I-1 to I-20, wherein the antibody is a full-length antibody.

Embodiment I-22. The antibody of any one of embodiments I-1 to I-20, wherein the antibody is an antibody fragment.

Embodiment I-23. The antibody of any one of embodiments I-1 to I-22, wherein the antibody is a humanized antibody.

Embodiment I-24. The antibody of any of embodiments I-1 to I-23, wherein the antibody contains a Fc-domain.

Embodiment I-25. The antibody of embodiment I-24, wherein the Fc domain is human.

Embodiment I-26. The antibody of embodiment I-25, wherein the Fc domain is human IgG1, IgG2, IgG3, or IgG4.

Embodiment I-27. The antibody of embodiment I-25, wherein the Fc domain is human IgG4.

Embodiment I-28. The antibody of embodiment I-27, wherein the Fc domain of the antibody is human IgG4, optionally SEQ ID NO: 139 and comprises at least one amino acid substitution at a position selected from the group consisting of: 215, 221, 222, 228, 234, 235, 236, 239, 240, 241, 243, 244, 245, 247, 250, 252, 254, 256, 262, 263, 264, 265, 266, 267, 268, 269, 270, 292, 296, 297, 298, 299, 300, 305, 313, 324, 325, 326, 327, 328, 329, 330, 332, 333, 334, 345, 396, 428, 430, 433, 434, and 440, or comprises one or more of the substitutions selected from the group consisting of T250Q/M428L, M252Y/S254T/T256E, M428L/N434S, S267E/L328F, N325S/L328F, and H433K/N434F, wherein the position numbers of the amino acid residues are of the EU numbering scheme.

Embodiment I-29. The antibody of any one of embodiments I-1 to I-22, wherein the antibody is a human antibody.

Embodiment I-30. The antibody of any one of embodiments I-1 to I-22, wherein the antibody is a chimeric antibody.

Embodiment 1-31. The antibody of any one of embodiments I-1 to I-30, wherein the antibody is conjugated.

Embodiment I-32. The antibody of embodiment 1-31, wherein the antibody is conjugated to a label.

Embodiment I-33. The antibody of any one of embodiments I-1 to I-32, wherein the antibody comprises any one or more of the amino acid sequences of the CDR sequences provided in Tables 1A, 1B, 1C, 2A, 2B, and 2C.

Embodiment I-34. The Protein S antibody of any one of embodiments I-1 to I-33, wherein the antibody comprises:

-   (a) any one of the CDR-L1 amino acid sequences of Table 1A; -   (b) any one of the CDR-L2 amino acid sequences of Table 1B; -   (c) any one of the CDR-L3 amino acid sequences of Table 1C; -   (d) any one of the CDR-H1 amino acid sequences of Table 2A; -   (e) any one of the CDR-H2 amino acid sequences of Table 2B; and -   (f) any one of the CDR-H3 amino acid sequences of Table 2B.

Embodiment I-35. The Protein S antibody of any one of embodiments I-1 to I-34, wherein the light chain variable domain of the antibody comprises:

-   (a) a CDR-L1 comprising the amino acid sequence of any one of SEQ ID     NOs: 7, 8, and 132; -   (b) a CDR-L2 comprising the amino acid sequence of any one of SEQ ID     NOs: 17 and 118; and -   (c) a CDR-L3 comprising the amino acid sequence of any one of SEQ ID     NOs: 28, 29, and 129.

Embodiment I-36. The Protein S antibody of any one of embodiments I-1 to I-35, wherein the heavy chain variable domain of antibody comprises:

-   (a) a CDR-H1 comprising the amino acid sequence of any one of SEQ ID     NOs: 40, 41, and 133; -   (b) a CDR-H2 comprising the amino acid sequence of any one of SEQ ID     NOs: 52, 53, and 134; and -   (c) a CDR-H3 comprising the amino acid sequence of any one of SEQ ID     NOs: 64, 65, and 135.

Embodiment I-37. The Protein S antibody of any one of embodiments I-1 to I-36, wherein the light chain variable domain of the antibody comprises:

-   (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; -   (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17;     and -   (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

Embodiment I-38. The Protein S antibody of any one of embodiments I-1 to I-36, wherein the light chain variable domain of the antibody comprises:

-   (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8; -   (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17;     and -   (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 29.

Embodiment I-39. The Protein S antibody of any one of embodiments I-1 to I-36, wherein the light chain variable domain of the antibody comprises:

-   (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 132; -   (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 118;     and -   (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 129.

Embodiment I-40. The Protein S antibody of any one of embodiments I-1 to I-39, wherein the heavy chain variable domain of the antibody comprises:

-   (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 40; -   (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52;     and -   (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 64.

Embodiment I-41. The Protein S antibody of any one of embodiments I-1 to I-39, wherein the heavy chain variable domain of the antibody comprises:

-   (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 41; -   (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 53;     and -   (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 65.

Embodiment I-42. The Protein S antibody of any one of embodiments I-1 to I-39, wherein the heavy chain variable domain of the antibody comprises:

-   (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 133; -   (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 134;     and -   (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 135.

Embodiment I-43. The Protein S antibody of any one of embodiments I-1 to I-32, wherein the antibody comprises the light and heavy CDR amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 52, and SEQ ID NO: 64.

Embodiment I-44. The Protein S antibody of any one of embodiments I-1 to I-32, wherein the antibody comprises the light and heavy CDR amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 41, SEQ ID NO: 53, and SEQ ID NO: 65.

Embodiment I-45. The Protein S antibody of any one of embodiments I-1 to I-32, wherein the antibody comprises the light and heavy CDR amino acid sequences of SEQ ID NO: 132, SEQ ID NO: 118, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135.

Embodiment I-46. The Protein S antibody of any one of embodiments I-1 to I-32, wherein the antibody comprises the variable chain amino acid sequence of any one of the amino acid sequences provided in Table 4A, and/or the variable chain amino acid sequence of any one of the amino acid sequences provided in Table 4B.

Embodiment I-47. The Protein S antibody of any one of embodiments I-1 to I-32, wherein the antibody comprises the light and heavy variable chain amino acid sequence of any one of the amino acid sequence combinations provided in Table 4C.

Embodiment I-48. The Protein S antibody of any one of embodiments I-1 to I-32, wherein the light chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 83 or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 84, or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

Embodiment I-49. The Protein S antibody of any one of embodiments I-1 to I-32, wherein the light chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 86, or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

Embodiment I-50. The Protein S antibody of any one of embodiments I-1 to I-32, wherein the light chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 155, or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

Embodiment I-51. The Protein S antibody of any one of embodiments I-1 to I-50, wherein the antibody comprises a light chain constant region, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 214.

Embodiment I-52. The Protein S antibody of any one of embodiments I-1 to I-50, wherein the antibody comprises a light chain constant region, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 215.

Embodiment I-53. The Protein S antibody of any one of embodiments I-1 to I-52, wherein, wherein the affinity for Protein S is about 0.001 nM or lower, 0.005 nM or lower, 0.01 nM or lower, 0.05 nM or lower, about 0.1 nM or lower, about 0.5 nM or lower, about 1 nM or lower, about 5 nM or lower, about 10 nM or lower, or about 50 nM or lower.

Embodiment I-54. A pharmaceutical composition comprising any one of the antibodies of embodiments I-1 to 1-53, and optionally a pharmaceutically acceptable excipient.

Embodiment I-55. A complex comprising Protein S and the antibody of any one of embodiments I-1 to 1-53, wherein the antibody is bound to the Protein S, and the Protein S is free.

Embodiment I-56. A complex comprising Protein S and the antibody of any one of embodiments I-1 to 1-53, wherein the antibody is bound to the Protein S, and the Protein S is complexed.

Embodiment I-57. The complex of embodiment I-56, wherein the Protein S is bound to C4BP.

Embodiment I-58. The complex of embodiment I-56, wherein the Protein S is bound to TFPI.

Embodiment I-59. A nucleic acid encoding for any one of the antibodies of embodiments I-1 to I-53.

Embodiment I-60. The nucleic acid of embodiment I-59, comprising any one of the nucleic acid sequences selected from Table 5A.

Embodiment I-61. The nucleic acid of any one of embodiments I-59 to I-60, comprising any one of the nucleic acid sequences selected from Table 5B.

Embodiment I-62. The nucleic acid of any one of embodiments I-59 to I-61, wherein the nucleic acid comprises the nucleic acid sequence of any one of the nucleic acid sequence combinations provided in Table 5C.

Embodiment I-63. The nucleic acid of any one of embodiments I-59 to I-62, wherein the light chain variable domain of a Protein S antibody of any one of embodiments I-1 to I-36 is encoded by the nucleic acid sequence of SEQ ID NO: 107 or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 108, or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

Embodiment I-64. The nucleic acid of any one of embodiments I-59 to I-62, wherein the light chain variable domain of a Protein S antibody of any one of embodiments I-1 to I-36 is encoded by the nucleic acid sequence of SEQ ID NO: 109 or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 110, or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

Embodiment I-65. The nucleic acid of any one of embodiments I-59 to I-62, wherein the light chain variable domain of a Protein S antibody of any one of embodiments I-1 to I-36 is encoded by the nucleic acid sequence of SEQ ID NO: 168 or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 169, or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

Embodiment I-66. A vector comprising the nucleic acid of any one of embodiments I-59 to I-65.

Embodiment I-67. An in vitro method of promoting the coagulation of blood, disrupting a Protein S-TFPI complex, or preventing the formation of a Protein S-TFPI complex, the method comprising contacting the antibody of any one of embodiments I-1 to I-53 with a blood sample.

Embodiment I-68. The method of embodiment I-67, wherein the blood sample comprises plasma.

Embodiment I-69. The method of any one of embodiments I-67 to I-68, wherein a marker associated with coagulation activity is increased.

Embodiment I-70. The method of any one of embodiments I-67 to I-69, wherein thrombin generation is promoted.

Embodiment I-71. The method of any one of embodiments I-67 to I-70, wherein fibrin generation is promoted.

Embodiment I-72. The method of any one of embodiments I-67 to I-71, wherein D-dimer levels are promoted.

Embodiment I-73. The method of any one of embodiments I-67 to I-72, wherein the blood sample is obtained from a subject having a coagulation factor deficiency, von Willebrand disease, or a platelet disorder.

Embodiment I-74. A method of promoting the coagulation of blood in a subject in need thereof, comprising administering to the subject the antibody of any one of embodiments I-1 to I-53 or the pharmaceutical composition of embodiment I-54.

Embodiment I-75. A method of promoting the generation of thrombin in a subject in need thereof, comprising administering to the subject the antibody of any one of embodiments I-1 to I-53 or the pharmaceutical composition of embodiment I-54.

Embodiment I-76. A method of decreasing circulating TFPI levels in the blood of a subject in need thereof, comprising administering to the subject the antibody of any one of embodiments I-1 to I-53 or the pharmaceutical composition of embodiment I-54.

Embodiment I-77. The method of embodiment I-76, wherein there is at least a 10% reduction in the circulating TFPI levels.

Embodiment I-78. The method of any one of embodiments I-76 to I-77, wherein the antibody disrupts TFPI-Protein S complexes in the subject.

Embodiment I-79. The method of any one of embodiments I-76 to I-78, wherein the antibody prevents the formation of TFPI-Protein S complexes in the subject.

Embodiment I-80. The method of any one of embodiments I-74 to I-79, wherein a marker associated with coagulation activity is increased.

Embodiment I-81. The method of any one of embodiments I-74 to I-80, wherein thrombin generation is promoted in the subject.

Embodiment I-82. The method of any one of embodiments I-74 to I-81, wherein fibrin generation is promoted in the subject.

Embodiment I-83. The method of any one of embodiments I-74 to I-82, wherein D-dimer levels are increased in the subject.

Embodiment I-84. The method of any one of embodiments I-74 to I-83, wherein the antibody remains active in the subject for a period of time.

Embodiment I-85. The method of any one of embodiments I-74 to I-84, wherein activity of the antibody is dose-dependent.

Embodiment I-86. The method of any one of embodiments I-74 to I-85, wherein the subject has a coagulation factor deficiency, von Willebrand disease, or a platelet disorder, and the antibody restores or promotes the generation of thrombin.

Embodiment I-87. The method of any one of embodiments I-74 to I-86, wherein the administration of the antibody is a subcutaneous administration.

Embodiment I-88. A method of treating a condition in a subject in need thereof, comprising administering to the subject the antibody of 1-53 or the pharmaceutical composition of embodiment I-54, wherein the condition is selected from the group consisting of: bleeding disorders, platelet disorders, trauma, and bleeding resulting from a surgery or a medical procedure.

Embodiment I-89. The method of embodiment I-88, wherein the method of treating is prophylactic.

Embodiment I-90. The method of embodiment I-88, wherein the method of treating is on-demand.

Embodiment I-91. The method of any one of embodiments I-88 to I-90, wherein the method is prophylactic and on-demand.

Embodiment I-92. The method of any one of embodiment I-89 or I-91, wherein the prophylactic method of treating is a routine prophylaxis.

Embodiment I-93. The method of any one of embodiments I-88 to I-92, wherein the administration of the antibody is a subcutaneous administration.

Embodiment I-94. The method of any one of embodiments I-74 to I-93, wherein the method of treating is acute.

Embodiment I-95. The method of any one of embodiments I-74 to I-93, wherein the method of treating is chronic.

Embodiment I-96. The method of any one of embodiments I-74 to I-93, wherein the method of treating is perioperative.

Embodiment I-97. The method of any one of embodiments I-74 to I-93, wherein the method of treating is intermittent.

Embodiment I-98. The method of any one of embodiments I-74 to I-97, wherein the antibody exhibits graded inhibition.

Embodiment I-99. The method of any one of embodiments I-74 to I-97, wherein the antibody exhibits switch-like inhibition.

Embodiment I-100. The method of any one of embodiments I-88 to I-99, wherein the subject suffers from two or more conditions selected from the group consisting of: bleeding disorders, platelet disorders, trauma, and bleeding resulting from a surgery or a medical procedure.

Embodiment I-101. The method of any one of embodiments I-86 to I-100, wherein the subject suffers from a bleeding disorder selected from the group consisting of: hemophilia A, hemophilia B, von Willebrand disease (vWD) disease, menorrhagia, Factor I deficiency, Factor II deficiency, Factor V deficiency, Factor VII deficiency, Factor X deficiency, Factor XI deficiency, Factor VIII deficiency (hemophilia A), Factor IX deficiency (hemophilia B), trauma, and hereditary hemorrhagic telangiectasia.

Embodiment I-102. The method of any one of embodiments I-74 to I-101, wherein the subject is a hemophilia carrier.

Embodiment I-103. The method of any one of embodiments I-75 to I-102, wherein the subject suffers from menorrhagia.

Embodiment I-104. The method of any one of embodiments I-74 to I-103, wherein the subject suffers from menorrhagia associated with one or more of the bleeding disorders or the platelet disorders.

Embodiment I-105. The method of embodiment I-101,wherein the bleeding disorder is vWD, and wherein the subject is undergoing a prophylactic treatment.

Embodiment I-106. The method of embodiment I-101,wherein the bleeding disorder is vWD, and the vWD is a subtype selected from: vWD Type 1, vWD Type 2A, vWD Type 2B, vWD Type 2N, vWD Type 2M, vWD Type 3, and acquired vWD.

Embodiment I-107. The method of any one of embodiments I-88 to I-106, wherein the condition is a platelet disorder selected from the group consisting of: Bernard-Soulier syndrome, Glanzmann’s thrombasthenia, and platelet storage pool deficiency.

Embodiment I-108. The method of embodiment I-107, wherein the platelet disorder is a platelet storage pool deficiency selected from the group consisting of: Gray platelet syndrome, Quebec platelet disorder, and MYH9-related thrombocytopenia (MYH9RD).

Embodiment I-109. The method of embodiment I-101,wherein the bleeding disorder is selected from Factor I deficiency, Factor II deficiency, Factor V deficiency, Factor VII deficiency, Factor X deficiency, Factor XI deficiency, Factor VIII deficiency (hemophilia A), Factor IX deficiency (hemophilia B), and vWD disease, and wherein the subject has inhibitors.

Embodiment I-110. The method of any one of embodiments I-88 to I-109, wherein the antibody or the pharmaceutical composition is capable of promoting thrombin generation in the subject.

Embodiment I-111. The method of any one of embodiments I-75 to I-86, wherein the thrombin generation does not exceed a predetermined threshold level.

Embodiment I-112. The method of any one of embodiments I-75 to I-86 and I-111, wherein the thrombin generation is antibody concentration-dependent.

Embodiment I-113. A kit or article of manufacture comprising an antibody of any one of embodiments I-1 to I-53 or the pharmaceutical composition of embodiment I-54.

Embodiment I-114. Use of the antibody of any one of embodiments I-1 to I-53 or the pharmaceutical composition of embodiment I-54 for the treatment of a condition in a subject in need thereof.

Embodiment I-115. Use of the antibody of any one of embodiments I-1 to I-53 or the pharmaceutical composition of embodiment I-54 for the manufacture of a medicament for the treatment of a condition in a subject in need thereof.

The present invention is not limited in scope by the specific embodiments described herein, which are intended as illustrations of individual aspects or embodiments of the invention. Functionally equivalent methods and components are within the scope of the invention. Various modifications of the invention, in addition to those described here, are apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications fall within the scope of the invention.

The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the antibodies of the present invention and practice the claimed methods. The following examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

EXAMPLES Example 1: Generation of Protein S Antibodies

The Protein S antibodies of the disclosure were generated as follows. Animals (e.g., mice, rats) were immunized with a full-length purified human plasma Protein S. Three immunization campaigns were carried out and standard techniques were used to generate hybridoma libraries from the animals. Flow cytometry and single cell sorting was used to generate single cell clones. Supernatants from these single clones were then screened for binding to both human and cynomolgus monkey Protein S. Clones that exhibited binding to both human and cynomolgus monkey Protein S were selected for expanded growth. The expanded cultures were then purified over Protein G or Protein A Sepharose using standard techniques. These purified antibody preparations were used in subsequent functional assays. The selected Protein S antibodies had a human variable region and a rat Fc domain.

Antibodies having a human variable region and a rat Fc domain (parental rat antibody) were made into fully human antibodies maintaining the same human variable domain as the parent antibody, with a human IgG4 Fc domain. Table 6 lists the Antibody number used to designate the human antibodies in the first column and the corresponding parental rat antibodies (with the human variable region) in the second column. The subsequent columns provide the variable light chain/variable heavy chain amino acid sequences and nucleic acid sequences, and the last column provides the amino acid sequences of the set of six CDRs that map to each antibody.

TABLE 6 Human Antibody: Protein S Antibody with Human Fc Rat Antibody: Protein S Antibody with Rat Fc Variable Light Chain, Variable Heavy Chain (Amino Acid Sequences) Variable Light Chain, Variable Heavy Chain (Nucleic Acid Sequences) CDR Combination (Amino Acid Sequences) Antibody 5 Antibody 17 SEQ ID NO: 83, SEQ ID NO: 84 SEQ ID NO: 107, SEQ ID NO: 108 SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 52, SEQ ID NO: 64 Antibody 10 Antibody 22 SEQ ID NO: 85, SEQ ID NO: 86 SEQ ID NO: 109, SEQ ID NO: 110 SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 41, SEQ ID NO: 53, SEQ ID NO: 65 Antibody 26 Antibody 32 SEQ ID NO: 154, SEQ ID NO: 155 SEQ ID NO: 168, SEQ ID NO: 169 SEQ ID NO: 132, SEQ ID NO: 118, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135

Example 2: Assessing Thrombin Generation in APC and TFPI Cofactor Activity Screening Assays Assessing APC and TFPI Cofactor Activity With Screening Assays

FIGS. 2A-2B depict exemplary assays performed to assess APC and TFPI cofactor activity, respectively and demonstrate how a TFPI cofactor inhibitor would behave in the assay.

The Thrombin Generation Assay (TGA) was used in this example, and was performed using a Thermo Fluoroskan Ascent Microplate Fluorometer and Thrombinoscope software. The PPP reagent (mixture of phospholipids and Tissue Factor) (Diagnostica Stago) was used in this example.

For the APC cofactor activity screening assay depicted in FIG. 2A, the following were tested: Protein S depleted platelet poor plasma, Protein S depleted plasma reconstituted with a Protein S and APC mixture, and Protein S depleted plasma reconstituted with a Protein S and APC mixture in the presence of varying concentrations of an anti-Protein S monoclonal antibody (mAb) known to display APC cofactor inhibitor activity. The Protein S and APC mixture was made by pre-mixing Protein S and APC and adding to the Protein S depleted plasma to create a Protein S-dependent assay, due to APC not showing inhibition of thrombin generation in the absence of Protein S. As shown, the addition of the pre-mixed Protein S and APC mixture to Protein S depleted plasma shows significantly less tissue factor-induced thrombin generation than the Protein S depleted plasma alone. Addition of anti-Protein S antibody to the Protein S depleted plasma + Protein S and APC mixture restored tissue factor-induced thrombin generation in a concentration-dependent manner.

For the TFPI cofactor activity screening assay depicted in FIG. 2B, the following were tested: platelet rich plasma with no treatment (also referred to herein as platelet-rich plasma, or PRP), a commercial Factor VIII (FVIII) neutralizing antibody, and a FVIII antibody and Protein S polyclonal antibody (pAB) mixture. The FVIII antibody acts as a neutralizing antibody and inhibits tissue factor-induced thrombin generation. Addition of the neutralizing Protein S antibodies enhanced thrombin generation in the presence of the FVIII antibody. Neutralizing Protein C antibodies did not show an effect on thrombin generation in the TFPI cofactor activity assay, indicating that endogenous Protein C or activated Protein C does not inhibit thrombin generation in this assay.

Example 3: Characterization of Selected Protein S Antibodies Characterization of Inhibitor Type by Cofactor Activity Assays

As a general matter, Protein S antibodies can be identified as either a dual inhibitor of Protein S cofactor activity for both APC and TFPI, for APC only (referred to interchangeably as APC cofactor inhibitor, APC cofactor specific inhibitor or APC pathway inhibitor), or for TFPI only (referred to interchangeably as TFPI cofactor inhibitor, TFPI cofactor specific inhibitor or TFPI pathway inhibitor) . The antibodies can be categorized by assessment of the thrombin generation profiles shown when assaying each antibody using the cofactor activity assays described in FIGS. 2A-2B.

Characterization of Protein S Antibodies

The results of the assays depicted in FIGS. 3A-6F were used to characterize the exemplary Protein S antibodies provided herein. Binding regions and calcium dependence were also determined. The results of the characterization assays are summarized in Tables 7 and 8 below and described in further detail in the below sections. The antibodies tested were determined to be TFPI cofactor inhibitors.

TABLE 7 Protein S Antibody Fc Pathway Inhibitor (APC, TFPI, or Dual) IC50 APC (nM) Hill Coefficie nt IC50 TFPI (nM) Hill Coefficie nt Antibody 22 Rat TFPI N/A N/A 4.4E-09 7.1 Antibody 17 Rat TFPI N/A N/A 1.033E-08 1.86 Antibody 32 Rat TFPI N/A N/A 5.02E-09 1.99

TABLE 8 Protein S Antibody with Rat Fc Pathway Inhibitor Binds Heavy Chain Calcium-Dependent Binding Binds Thrombin-Sensitive Region of Protein S Antibody 22 TFPI No No No Antibody 17 TFPI No No No Antibody 32 TFPI No No No

Binding Characteristics of Protein S Antibodies

Binding affinities of the Protein S antibodies provided herein were also determined.

Binding interaction analysis was obtained by Surface Plasmon Resonance in a Biacore X100 instrument. The human anti-IgG (Fc) antibody was immobilized to the carboxymethylated dextran membrane on the sensor chip surface (CM5) via the free amine method for a contact time of 420 seconds. Human anti-IgG antibody at 25 µg/mL in 0.15 M NaCl was immobilized to 9,785 RU in 10 mM sodium acetate pH 5.0. Each tested monoclonal antibody was captured at a fixed concentration (0.25 µg/mL) with immobilized human anti-IgG antibody. Experimentally, the capture ligand level for the antibodies tested was determined to be 66-99 RL, corresponding to a R_(max) of 61-91 RU. No signs of mass transport limitation were observed.

The following experimental conditions were used: purified Protein S was injected at concentrations: 2-128 nM, 1:2-fold dilution. Dilution and running buffer were as follows: Hbs-EP + 5 mM CaCl₂. Regeneration was obtained by 3 M MgCl₂. Binding constants (ka, kd, and KD) were determined using the Biacore X100 evaluation software, assuming a 1:1 interaction of Protein S and the tested monoclonal antibody under investigation. The resulting data are presented in Table 9A below. Antibody 5 and 10 show a KD of binding of less than 50 nM in the Biacore assay.

TABLE 9A Ka (1/MS) Kd (1/S) KD nM Antibody 5 8.12E+04 2.27E-03 28 Antibody 10 1.67E+05 5.14E-03 31

Binding affinities of the Protein S antibodies provided herein were also further characterized using the Octet System (Sartorius). The binding of each antibody to both human and cynomolgus monkey (“cyno”) Protein S was determined. The human Fc antibodies were immobilized onto anti-human Fc capture probes by placing the probes into 10 µg/ml antibody solution in 10 mg/ml bovine serum albumin, 20 mM Tris pH 7.0, 150 mM NaCl, and 4 mM calcium chloride. Next, the bound antibodies were placed into solutions containing 500 nM, 250 nM, 125 nM 62.5 nM, 31.25 nM, 15.625 nM and 7.81 nM Protein S and the association rates were measured. Next, the probes were placed into buffer and the dissociation rates were measured. A summary of the resulting data is provided in Table 9B below. Antibody 5 shows a KD of less than 10 nM in the Octet system. Antibody 10 shows a KD of less than 50 nM in the Octet system. Antibody 26 shows a KD of less than 0.5 nM in the Octet system.

TABLE 9B Antibody k_(on) (1/MS) kdis (1/s) KD (M) (human) k_(on) (1/MS) kdis (1/s) KD (M) (cyno) Antibody 5 6.33E+04 4.93E-04 7.80E-09 6.75E+04 2.83E-04 4.19E-09 Antibody 10 1.27E+05 2.13E-03 1.68E-08 1.07E+05 1.32E-03 1.23E-08 Antibody 26 9.85E+04 4.58E-05 4.66E-10 9.03E+04 3.15E-05 3.49E-10

Binding to the heavy chain of Protein S was determined using an ELISA based method. Briefly, high binding ELISA plates were coated with recombinant heavy chain Protein S expressed and purified from HEK293 cells. The “heavy chain” of Protein S represents a fragment of Protein S spanning amino acids 42-296. The coated plate was blocked with 1% casein solution. Then, buffer containing 1 µg/ml of antibody to be tested was applied to the well with buffer containing 1 mM calcium chloride. Table 10 below summarizes the results of the heavy chain binding assay for all Antibodies. The tested antibodies did not exhibit any significant binding to the heavy chain of Protein S. (antibodies that do bind this region have ODs greater than about 0.5 OD).

TABLE 10 Binding to heavy chain (OD) Antibody 17 0.07 Antibody 22 0.06 Antibody 32 0.10

Calcium dependence of binding was determined using an ELISA based method. Briefly, high binding ELISA plates were coated with human Protein S. Then, buffer containing 1 µg/ml of antibody was applied to the well with either buffer containing 1 mM EDTA or 1 mM calcium chloride. If the level of binding of Protein S was reduced (greater than 85% of the absorbance observed in the presence of calcium) when EDTA was added, it was concluded that the binding was calcium-dependent. Table 11 summarizes the results of the calcium dependence assay for all antibodies. The tested antibodies did not exhibit calcium-dependent binding.

TABLE 11 Binding to Protein S in EDTA (OD) Binding to Protein S in CaCl₂ (OD) Antibody 17 0.39 0.54 Antibody 22 0.07 0.20 Antibody 32 0.33 0.82

Antibody 17 and Antibody 5

FIGS. 3A-3G depict the characterization of Antibody 17 and Antibody 5, antibodies sharing the same human variable region, and are characterized as TFPI cofactor inhibitors.

Binding to the heavy chain of Protein S was determined using an ELISA based method, as described herein. Antibody 17 was determined to not bind to the heavy chain of Protein S. Calcium dependence was determined, also as described herein, and Antibody 17 showed binding that was calcium-independent.

FIGS. 3A-3C depict the results of an APC cofactor assay for Antibody 17 using Protein S deficient human PPP, cynomolgus monkey PPP, and a TFPI cofactor assay using human PRP, respectively.

FIGS. 3D and 3E depict results of a TFPI cofactor assay in human PRP using Antibody 5 and controls, respectively. A rabbit polyclonal human Protein S antibody labeled Dako was used as a positive control.

FIGS. 3F and 3G depict the results of the APC cofactor assay performed with Antibody 5 and controls, respectively.

These results indicate that Antibody 17 and Antibody 5 are TFPI cofactor inhibitors.

Antibody 22 and Antibody 10

FIGS. 4A-4E depict the characterization of Antibody 22 and Antibody 10, antibodies sharing the same human variable region, and are characterized as TFPI cofactor specific inhibitors.

Binding to the heavy chain of Protein S was determined using an ELISA based method, as described herein. Antibody 22 was determined to not bind to the heavy chain of Protein S. Calcium dependence was determined, also as described herein, and Antibody 22 showed binding that was calcium-independent.

FIGS. 4A-4C depict the results of a APC cofactor assay for Antibody 22 using Protein S deficient human PPP, cynomolgus monkey PPP, and a TFPI cofactor assay using human PRP, respectively.

FIGS. 4D and 4E depict the results of a TFPI cofactor assay in human PRP using Antibody 10 and controls, respectively.

These results indicate that Antibody 22 and Antibody 10 are TFPI cofactor inhibitors.

Antibody 18

FIG. 4F depicts the characterization of Antibody 26, and is characterized as a TFPI cofactor inhibitor. FIG. 4F depicts the results of a TFPI cofactor assay in human PRP using Antibody 26.

These results indicate that Antibody 26 is a TFPI cofactor specific inhibitor.

Example 4: Binding of Protein S Antibodies to the Protein S-C4BP Complex

The Octet assay was used to determine the capability of the Protein S antibodies provided herein to bind to Protein S either alone, or when complexed to C4BP. A summary of the binding to Protein S or the Protein S-C4BP complex is provided in Table 12. Table 12 presents an X where a tested antibody was able to bind to either the Protein S-C4BP Complex, and/or the Protein S alone. A dash indicates that the binding did not occur.

Using the Octet System (Sartorius), the binding of each antibody to both Protein S and Protein S in complex with C4BP was determined. It had previously been determined that the C4BP preparation from Complement Technologies contains Protein S precomplexed to human C4BP. Therefore, this preparation represented the Protein S-C4BP complex and plasma purified Protein S from Haematologic Technologies was used as the source of free Protein S. The human Fc antibodies were immobilized onto anti-human Fc capture probes by placing the probes into 10 µg/ml antibody solution in 10 mg/ml bovine serum albumin, 20 mM Tris pH 7.0, 150 mM NaCl, and 4 mM calcium chloride. Then the bound antibodies were placed into solutions containing either 75 µg/ml Protein S or 100 µg/ml C4BP (C4BP-Protein S complex). With antibodies that do not bind Protein S in complex with C4BP, no binding is observed. With antibodies that bind Protein S in free form or in complex with C4BP, an association is observed under both conditions. All antibodies tested bound free Protein S. Antibody 5 showed the ability to bind Protein S when in a complex with C4BP.

TABLE 12 Antibody with Human Fc Binding to ProS-C4BP Complex Binding to ProS Antibody 5 X X Antibody 10 - X Antibody 26 - X

Example 5: Effects of Protein S Antibodies on Fibrin Deposition

Fibrin deposition experiments were carried out as follows. Bioflux 1000z 48-well high shear microfluidics plates (0-200 dynes/cm2) employed in the experiments were purchased from Bioflux. The device has the #1.5 borosilicate glass coverslip which forms the floor of the microfluidic channel and is engineered to facilitate the coating of collagen. Collagen type 1 was purchase from Chrono-Par collagen (chrono-Log Corp, Havertown, PA). Collagen was perfused from the wells at room temperature and incubated for 1 hour. Precoated plates with collagen type 1 were rinsed with PBS and channels were blocked with 0.5% (v/v) BSA for 10 min in PBS prior to the addition of the labeled blood to the wells. Sodium citrate anti-coagulated whole human blood (ALLCells, Oakland, CA) was used within 4-8h of collection. Whole blood was incubated for 1 hour with 100 µg/mL Sheep anti-Human Factor VIII (Haematologic Technologies, Essex, VT). VGA antibodies were added and incubated with whole blood. Fibrinogen from human plasma, Alexa Fluor 546 (Invitrogen, Carlsbad, CA) was added at a final concentration of 50 µg/mL prior to biological experiments. Whole blood was added to the input wells and perfused at 30 dyn/cm2 using the BioFlux Controller and software. The samples were illuminated for no more than 30 ms for each capture. The BioFlux software imaging module was used to control the image acquisition settings and to process the fluorescence intensity measurements. Fluorescent micrographs were captured with the blood under flow using an inverted microscope (ZEISS Axio Observer 7) and sCMOS Camera. Images were timelapse recorded using the BioFlux 1000 imaging system (Fluxion Biosciences). Data was processed using BioFlux Montage Software.

FIGS. 5A-5B depict the level of fibrin deposition resulting from the Protein S antibodies added to whole blood treated with Factor VIII neutralizing antibodies. This experiment tests the ability of the Protein S antibodies to restore or increase fibrin deposition in an in vitro microfluidic Hemophilia A model. Whole blood was treated with Factor VIII antibody to decrease fibrin deposition. FIG. 5A depicts the effects of Antibody 10, and a control on fibrin deposition, and FIG. 5B depicts the effects of Antibody 5 and a control on fibrin deposition in anti-FVIII treated human plasma. These results demonstrate that the Protein S antibodies 5 and 10 could restore or increase fibrin deposition in an in vitro microfluidic Hemophilia A model.

Example 6: Effect of Protein S Antibodies in an in Vitro Microfluidics Hemophilia A Bleeding Model

These experiments were carried out using a fully endothelialized microfluidic system that was coupled to a microengineered pneumatic valve that mimics vascular damage (Sakurai, et al. Nature Communications 2018). Briefly, whole blood collected from healthy volunteers were treated with a sheep anti-human FVIII antibody to mimic whole blood from a hemophilia A patient. After treatment, the whole blood was perfused into the microfluidics system at which time an “injury” was introduced using the pneumatic valve. The localization of both platelets and fibrin at the site of “injury” was monitored and the time to cessation of “bleeding” was measured.

FIGS. 6A-6B depict the effects of Protein S antibodies in an in vitro microfluidics Hemophilia A bleeding model, measured by microfluidics experiments. These experiments were performed to measure the time of various Protein S antibodies to stop bleeding, and to determine the extent to which these antibodies induced fibrin deposition, respectively, in a microengineered, vascularized Hemophilia A bleeding model. The tested antibody was Antibody 5. These results demonstrate that the antibody was able to restore or increase fibrin deposition in a Hemophilia A bleeding model and therefore stop bleeding.

Example 7: Effect of Protein S Antibodies on Thrombin Generation

These assays were carried out as follows. The Thrombin Generation Assay (TGA) was performed using a Thermo Fluoroskan Ascent Microplate Fluorometer and Thrombinoscope software. The PPP low reagent (Diagnostica Stago) was used in this experiment. Briefly, plasma from von Willebrand diseased patients was mixed with increasing levels of Protein S antibodies and incubated at room temperature. Then soluble human thrombomodulin was added or not added prior to initiation of the reaction. To start the reaction, PPP low reagent was added along with calcium and the thrombin substrate. The levels of thrombin were then monitored over time.

FIGS. 7A-7B depict results of enhanced dose-dependent thrombin generation when Protein S antibodies were added to plasma obtained from patients with various types of von Willebrand disease, in the absence or presence of exogenously added thrombomodulin, respectively. The figures depict enhanced thrombin generation when Antibody 5 was added to various samples of congenital factor deficient plasma. Significantly less thrombin generation was observed with thrombomodulin, a cofactor of thrombin that is involved in conversion of Protein C to activated Protein C, an anticoagulant enzyme. Plasma samples were taken from patients having von Willebrand disease (vWD) type 1, 2B, or 3.

Antibody 5 was able to effectively restore thrombin generation.

Example 8: Results From Cynomolgus Monkeys Receiving Subcutaneous and Intravenous Injections of Protein S Antibodies

This example depicts the results of ex vivo pharmacodynamic (PD) assays and ex vivo pharmacokinetic (PK) assays, used to measure the level of inhibition as well as the free antibody concentration, respectively, in cynomolgus monkeys injected subcutaneously or intravenously with Protein S antibodies of the disclosure. These results also depict levels of D-dimer, used as a marker of coagulation activity, observed in the monkeys. The assays were performed with plasma samples collected after administration of Protein S antibodies dosed in cynomolgus monkeys at 3 mg/kg subcutaneously and intravenously.

More specifically, to assess the pharmacodynamic effects of in vivo administered Protein S antibodies, a TGA was performed on plasma samples using a Thermo Fluoroskan Ascent Microplate Fluorometer and Thrombinoscope software. The PPP reagent (Diagnostica Stago) was used in this experiment. Briefly, cynomolgus monkey plasma was mixed with PPP reagent and 5 µg/ml of activated Protein C along with calcium and substrate and the levels of thrombin generation was monitored over 1 hour. The PK assay was performed by incubating diluted cynomolgus monkey plasma onto plates immobilized with human Protein S. The plates used were MSD 96-well plates. 30 µl of 2 µg/ml plasma purified Protein S in Tris buffer containing calcium was used to coat the plate overnight. After blocking, the wells were incubated with samples, standards and QCs. 25 µl per well of 2 µg/ml sulfo-tagged goat anti-human IgG, monkey was added and incubated at room temperature for 1 hr. After washing, 150 µl of 1X MSD Read Buffer T in water was added to each well and the plate was read on a MSD plate reader. The levels of D-dimer were measured using the D-dimer assay kit from Diagnostica Stago following manufacturer’s recommended protocol.

FIG. 8A depicts the levels of free antibody in plasma when Antibody 5 was administered to cynomolgus monkeys at 3 mg/kg subcutaneously or intravenously. FIG. 8B depicts levels of D-dimer, as a marker of coagulation activity, measured over time in cynomolgus monkeys injected with Antibody 5.

FIGS. 8C and 8D depict the levels of free antibody (left axis, filled circles) and percent inhibition of Protein S activity in the TGA (right axis, filled triangles) when Antibody 10 was administered to cynomolgus monkeys at 3 mg/kg subcutaneously and intravenously, respectively. FIG. 8E depicts levels of D-dimer, as a marker of coagulation activity, measured over time in cynomolgus monkeys injected with Antibody 10.

FIGS. 8F and 8G depict the levels of free antibody (left axis, filled squares for FIG. 8F; left axis, filled circles for FIG. 8G) and percent inhibition of Protein S activity in the TGA (right axis, filled triangles) when Antibody 26 was administered to cynomolgus monkeys at 3 mg/kg subcutaneously and intravenously, respectively. FIG. 8H depicts levels of D-dimer, as a marker of coagulation activity, measured over time in cynomolgus monkeys injected with Antibody 26.

Based on the results, the administration of antibodies 10 and 26 at 3 mg/kg injected subcutaneously or intravenously exhibited a pharmacodynamic effect for the duration of the study, which was 168 hours. The antibodies tested showed inhibition of Protein S activity as assayed in the TGA. Additionally, plasma samples also showed the increase of D-dimer levels over time, demonstrating the intended pharmacodynamic effect of increased activity of the coagulation system in Protein S antibody dosed monkeys.

Example 9: Effects of Protein S Antibodies on the Binding of Protein S to TFPI

The effect of the antibodies of the disclosure on the binding of Protein S to TFPI was measured.

Using the Octet System (Sartorius), an assay to measure the binding of TFPI to Protein S was developed. Briefly, the human Fc antibodies were immobilized onto anti-human Fc capture probes (anti-hIgG probe) by placing the probes into 10 µg/ml antibody solution in 10 mg/ml bovine serum albumin, 20 mM Tris pH 7.0, 150 mM NaCl, and 4 mM calcium chloride. Then the bound antibodies were placed into a solution containing 10 µg/ml human Protein S followed by a 10 µg/ml solution containing human TFPI. Finally, the probe was placed into a buffer solution (wash) to observe the dissociation of TFPI from Protein S. The kinetics of association and dissociation of TFPI to Protein S were measured. Each step of this assay is depicted in FIGS. 9A-9C.

FIG. 9A depicts the lack of binding of human TFPI to Protein S after the binding of Protein S to Antibody 5 (marked with a *). These results demonstrate that Antibody 5 prevents the formation of a TFPI-Protein S complex.

FIG. 9B depicts the lack of binding of human TFPI to Protein S after the binding of Protein S to Antibody 10 (marked with a *). These results demonstrate that Antibody 10 prevents the formation of a TFPI-Protein S complex.

FIG. 9C depicts the results obtained using a control antibody that does not block TFPI binding to Protein S (marked with a *). This control antibody, does not prevent the formation of a TFPI-Protein S complex.

These results support the use of the Protein S antibodies provided herein for depleting TFPI by preventing its association with Protein S, either by disrupting a TFPI-Protein S complex, or by preventing the formation of a TFPI-Protein S complex.

Example 10: Effect of Protein S Antibodies on TFPI Plasma Levels in Cynomolgus Monkeys

TFPI levels were measured using the Human TFPI Quantikine ELISA kit from R&D systems following the manufacturer’s suggested protocol. The cynomolgus monkey plasma was diluted 20-fold for the assay.

FIG. 10 depicts the decreasing TFPI levels in plasma of cynomolgus monkeys over time, following repeated 3 mg/kg subcutaneous administration of a Protein S antibody dosed at Times 0 hrs, 72 hrs and 144 hours. Briefly, Antibody 5 was administered to five monkeys, and the TFPI levels were measured over a period of approximately 175 hours post-dose. These results demonstrate that the TFPI levels were reduced over time following the repeated antibody administration, and that the disruption of the Protein S-TFPI interaction lowers plasma levels of TFPI.

FIG. 11 is TFPI ELISA data from plasma samples taken from cynomolgus monkeys dosed once at 3 mg/kg IV with Protein S antibodies. The data show that dosing with Antibodies 5, 10, and 26 induced a drop in circulating TFPI levels. Control antibodies with the same Fc, but a different antigen binding domain did not induce such a drop.

Data are not shown, but the dosing of at least Antibody 5 did not lead to a concomitant drop in Protein S levels.

Example 11: Effect of Protein S Antibodies on Thrombin Generation in a TFPI Cofactor Assay

Since TFPI and Protein S exist in blood as a protein complex, the treatment of whole blood with Protein S antibodies only allows for the examination of the ability of the antibody to inhibit Protein S wherein the Protein S is already in a complex with TFPI or allows for the examination of the disruption of a Protein S-TFPI complex. To examine whether certain Protein S antibodies could prevent the complexation of TFPI and Protein S, Protein S-depleted plasma was utilized. The Protein S depleted plasma was also depleted of TFPI.

Protein S was added to Protein S-depleted plasma along with either buffer (control) or increasing levels of Protein S antibody. Following the incubation, recombinant TFPI (R&D Systems), which lacks the last 20 amino acids at the C-terminus of TFPI, was added and the level of thrombin generation was measured. For Antibodies 5 and 10, a dose-dependent increase in thrombin generation was observed.

FIGS. 12A-12B depict the effects of different doses of Antibody 5 and Antibody 10 on thrombin levels in Protein S - depleted human plasma where Protein S (+/- antibody) and TFPI were added back to Protein S depleted plasma.

Based on the results of this TFPI cofactor assay, Antibody 5 and Antibody 10 can promote thrombin generation by preventing the association of Protein S with TFPI. 

1. An antibody that binds Protein S, wherein the binding of the antibody to Protein S decreases circulating tissue factor pathway inhibitor (TFPI) levels.
 2. An antibody that binds Protein S, wherein the binding of the antibody to Protein S disrupts a Protein S-TFPI complex, or prevents the formation of a Protein S-TFPI complex.
 3. The antibody of any one of claims 1-2, wherein the antibody is an inhibitor for the cofactor activity of Protein S for TFPI.
 4. The antibody of any one of claims 1-3, wherein the binding of the antibody to Protein S induces at least a 10% decrease in circulating TFPI levels.
 5. The antibody of any one of claims 1-4, wherein the binding of the antibody to Protein S disrupts the Protein S-TFPI complex.
 6. The antibody of any one of claims 1-5, wherein the binding of the antibody to Protein S prevents the formation of the Protein S-TFPI complex.
 7. The antibody of any one of claims 1-6, wherein the antibody binds to the C-terminus of Protein S.
 8. The antibody of any one of claims 1-7, wherein the antibody binds to an sex hormone binding globulin-like domain (SHBG-like domain) of Protein S.
 9. The antibody of any one of claims 1-8, wherein the binding of the antibody to Protein S promotes generation of a marker associated with coagulation activity.
 10. The antibody of any one of claims 1-9, wherein the binding of the antibody to Protein S promotes thrombin generation.
 11. The antibody of any one of claims 1-10, wherein the binding of the antibody to Protein S promotes D-dimer levels.
 12. The antibody of any one of claims 1-11, wherein the binding of the antibody to Protein S promotes fibrin generation.
 13. The antibody of any one of claims 1-12, wherein the activity of the antibody is dose-dependent.
 14. The antibody of any one of claims 1-13, wherein activity of the antibody is measured in vitro.
 15. The antibody of any one of claims 1-13, wherein activity of the antibody is measured in vivo.
 16. The antibody of any one of claims 1-15, wherein the antibody binds to free Protein S.
 17. The antibody of any one of claims 1-15, wherein the antibody binds to complexed Protein S.
 18. The antibody of claim 17, wherein the Protein S is complexed with C4BP.
 19. The antibody of claim 17, wherein the Protein S is complexed with TFPI.
 20. The antibody of any one of claims 1-19, wherein the antibody is a monoclonal antibody.
 21. The antibody of any one of claims 1-20, wherein the antibody is a full-length antibody.
 22. The antibody of any one of claims 1-20, wherein the antibody is an antibody fragment.
 23. The antibody of any one of claims 1-22, wherein the antibody is a humanized antibody.
 24. The antibody of any of claims 1-23, wherein the antibody contains a Fc-domain.
 25. The antibody of claim 24, wherein the Fc domain is human.
 26. The antibody of claim 25, wherein the Fc domain is human IgG1, IgG2, IgG3, or IgG4.
 27. The antibody of claim 25, wherein the Fc domain is human IgG4.
 28. The antibody of claim 27, wherein the Fc domain of the antibody is human IgG4, optionally SEQ ID NO: 139 and comprises at least one amino acid substitution at a position selected from the group consisting of: 215, 221, 222, 228, 234, 235, 236, 239, 240, 241, 243, 244, 245, 247, 250, 252, 254, 256, 262, 263, 264, 265, 266, 267, 268, 269, 270, 292, 296, 297, 298, 299, 300, 305, 313, 324, 325, 326, 327, 328, 329, 330, 332, 333, 334, 345, 396, 428, 430, 433, 434, and 440, or comprises one or more of the substitutions selected from the group consisting of T250Q/M428L, M252Y/S254T/T256E, M428L/N434S, S267E/L328F, N325S/L328F, and H433K/N434F, wherein the position numbers of the amino acid residues are of the EU numbering scheme.
 29. The antibody of any one of claims 1-22, wherein the antibody is a human antibody.
 30. The antibody of any one of claims 1-22, wherein the antibody is a chimeric antibody.
 31. The antibody of any one of claims 1-30, wherein the antibody is conjugated.
 32. The antibody of claim 31, wherein the antibody is conjugated to a label.
 33. The antibody of any one of claims 1-32, wherein the antibody comprises any one or more of the amino acid sequences of the CDR sequences provided in Tables 1A, 1B, 1C, 2A, 2B, and 2C.
 34. The Protein S antibody of any one of claims 1-33, wherein the antibody comprises: (a) any one of the CDR-L1 amino acid sequences of Table 1A; (b) any one of the CDR-L2 amino acid sequences of Table 1B; (c) any one of the CDR-L3 amino acid sequences of Table 1C; (d) any one of the CDR-H1 amino acid sequences of Table 2A; (e) any one of the CDR-H2 amino acid sequences of Table 2B; and (f) any one of the CDR-H3 amino acid sequences of Table 2B.
 35. The Protein S antibody of any one of claims 1-34, wherein the light chain variable domain of the antibody comprises: (a) a CDR-L1 comprising the amino acid sequence of any one of SEQ ID NOs: 7, 8, and 132; (b) a CDR-L2 comprising the amino acid sequence of any one of SEQ ID NOs: 17 and 118; and (c) a CDR-L3 comprising the amino acid sequence of any one of SEQ ID NOs: 28, 29, and
 129. 36. The Protein S antibody of any one of claims 1-35, wherein the heavy chain variable domain of antibody comprises: (a) a CDR-H1 comprising the amino acid sequence of any one of SEQ ID NOs: 40, 41, and 133; (b) a CDR-H2 comprising the amino acid sequence of any one of SEQ ID NOs: 52, 53, and 134; and (c) a CDR-H3 comprising the amino acid sequence of any one of SEQ ID NOs: 64, 65, and
 135. 37. The Protein S antibody of any one of claims 1-36, wherein the light chain variable domain of the antibody comprises: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:
 28. 38. The Protein S antibody of any one of claims 1-36, wherein the light chain variable domain of the antibody comprises: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:
 29. 39. The Protein S antibody of any one of claims 1-36, wherein the light chain variable domain of the antibody comprises: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 132; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 118; and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:
 129. 40. The Protein S antibody of any one of claims 1-39, wherein the heavy chain variable domain of the antibody comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 52; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:
 64. 41. The Protein S antibody of any one of claims 1-39, wherein the heavy chain variable domain of the antibody comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 41; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 53; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:
 65. 42. The Protein S antibody of any one of claims 1-39, wherein the heavy chain variable domain of the antibody comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 133; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 134; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:
 135. 43. The Protein S antibody of any one of claims 1-32, wherein the antibody comprises the light and heavy CDR amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 40, SEQ ID NO: 52, and SEQ ID NO:
 64. 44. The Protein S antibody of any one of claims 1-32, wherein the antibody comprises the light and heavy CDR amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 41, SEQ ID NO: 53, and SEQ ID NO:
 65. 45. The Protein S antibody of any one of claims 1-32, wherein the antibody comprises the light and heavy CDR amino acid sequences of SEQ ID NO: 132, SEQ ID NO: 118, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO:
 135. 46. The Protein S antibody of any one of claims 1-32, wherein the antibody comprises the variable chain amino acid sequence of any one of the amino acid sequences provided in Table 4A, and/or the variable chain amino acid sequence of any one of the amino acid sequences provided in Table 4B.
 47. The Protein S antibody of any one of claims 1-32, wherein the antibody comprises the light and heavy variable chain amino acid sequence of any one of the amino acid sequence combinations provided in Table 4C.
 48. The Protein S antibody of any one of claims 1-32, wherein the light chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 83 or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 84, or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
 49. The Protein S antibody of any one of claims 1-32, wherein the light chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 86, or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
 50. The Protein S antibody of any one of claims 1-32, wherein the light chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 154 or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody comprises the amino acid sequence of SEQ ID NO: 155, or an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
 51. The Protein S antibody of any one of claims 1-50, wherein the antibody comprises a light chain constant region, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:
 214. 52. The Protein S antibody of any one of claims 1-50, wherein the antibody comprises a light chain constant region, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:
 215. 53. The Protein S antibody of any one of claims 1-52, wherein, wherein the affinity for Protein S is about 0.001 nM or lower, 0.005 nM or lower, 0.01 nM or lower, 0.05 nM or lower, about 0.1 nM or lower, about 0.5 nM or lower, about 1 nM or lower, about 5 nM or lower, about 10 nM or lower, or about 50 nM or lower.
 54. A pharmaceutical composition comprising any one of the antibodies of claims 1-53, and optionally a pharmaceutically acceptable excipient.
 55. A complex comprising Protein S and the antibody of any one of claims 1-53, wherein the antibody is bound to the Protein S, and the Protein S is free.
 56. A complex comprising Protein S and the antibody of any one of claims 1-53, wherein the antibody is bound to the Protein S, and the Protein S is complexed.
 57. The complex of claim 56, wherein the Protein S is bound to C4BP.
 58. The complex of claim 56, wherein the Protein S is bound to TFPI.
 59. A nucleic acid encoding for any one of the antibodies of claims 1-53.
 60. The nucleic acid of claim 59, comprising any one of the nucleic acid sequences selected from Table 5A.
 61. The nucleic acid of any one of claims 59-60, comprising any one of the nucleic acid sequences selected from Table 5B.
 62. The nucleic acid of any one of claims 59-61, wherein the nucleic acid comprises the nucleic acid sequence of any one of the nucleic acid sequence combinations provided in Table 5C.
 63. The nucleic acid of any one of claims 59-62, wherein the light chain variable domain of a Protein S antibody of any one of claims 1-36 is encoded by the nucleic acid sequence of SEQ ID NO: 107 or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 108, or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
 64. The nucleic acid of any one of claims 59-62, wherein the light chain variable domain of a Protein S antibody of any one of claims 1-36 is encoded by the nucleic acid sequence of SEQ ID NO: 109 or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 110, or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
 65. The nucleic acid of any one of claims 59-62, wherein the light chain variable domain of a Protein S antibody of any one of claims 1-36 is encoded by the nucleic acid sequence of SEQ ID NO: 168 or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or wherein the heavy chain variable domain of the antibody is encoded by the nucleic acid sequence of SEQ ID NO: 169, or a nucleic acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
 66. A vector comprising the nucleic acid of any one of claims 59-65.
 67. An in vitro method of promoting the coagulation of blood, disrupting a Protein S-TFPI complex, or preventing the formation of a Protein S-TFPI complex, the method comprising contacting the antibody of any one of claims 1-53 with a blood sample.
 68. The method of claim 67, wherein the blood sample comprises plasma.
 69. The method of any one of claims 67-68, wherein a marker associated with coagulation activity is increased.
 70. The method of any one of claims 67-69, wherein thrombin generation is promoted.
 71. The method of any one of claims 67-70, wherein fibrin generation is promoted.
 72. The method of any one of claims 67-71, wherein D-dimer levels are promoted.
 73. The method of any one of claims 67-72, wherein the blood sample is obtained from a subject having a coagulation factor deficiency, von Willebrand disease, or a platelet disorder.
 74. A method of promoting the coagulation of blood in a subject in need thereof, comprising administering to the subject the antibody of any one of claims 1-53 or the pharmaceutical composition of claim
 54. 75. A method of promoting the generation of thrombin in a subject in need thereof, comprising administering to the subject the antibody of any one of claims 1-53 or the pharmaceutical composition of claim
 54. 76. A method of decreasing circulating TFPI levels in the blood of a subject in need thereof, comprising administering to the subject the antibody of any one of claims 1-53 or the pharmaceutical composition of claim
 54. 77. The method of claim 76, wherein there is at least a 10% reduction in the circulating TFPI levels.
 78. The method of any one of 76-77, wherein the antibody disrupts TFPI-Protein S complexes in the subject.
 79. The method of any one of 76-78, wherein the antibody prevents the formation of TFPI-Protein S complexes in the subject.
 80. The method of any one of claims 74-79, wherein a marker associated with coagulation activity is increased.
 81. The method of any one of claims 74-80, wherein thrombin generation is promoted in the subject.
 82. The method of any one of claims 74-81, wherein fibrin generation is promoted in the subject.
 83. The method of any one of claims 74-82, wherein D-dimer levels are increased in the subject.
 84. The method of any one of claims 74-83, wherein the antibody remains active in the subject for a period of time.
 85. The method of any one of claims 74-84, wherein activity of the antibody is dose-dependent.
 86. The method of any one of claims 74-85, wherein the subject has a coagulation factor deficiency, von Willebrand disease, or a platelet disorder, and the antibody restores or promotes the generation of thrombin.
 87. The method of any one of claims 74-86, wherein the administration of the antibody is a subcutaneous administration.
 88. A method of treating a condition in a subject in need thereof, comprising administering to the subject the antibody of 1-53 or the pharmaceutical composition of claim 54, wherein the condition is selected from the group consisting of: bleeding disorders, platelet disorders, trauma, and bleeding resulting from a surgery or a medical procedure.
 89. The method of claim 88, wherein the method of treating is prophylactic.
 90. The method of claim 88, wherein the method of treating is on-demand.
 91. The method of any one of claims 88-90, wherein the method is prophylactic and on-demand.
 92. The method of any one of claims 89 or 91, wherein the prophylactic method of treating is a routine prophylaxis.
 93. The method of any one of claims 88-92, wherein the administration of the antibody is a subcutaneous administration.
 94. The method of any one of claims 74-93, wherein the method of treating is acute.
 95. The method of any one of claims 74-93, wherein the method of treating is chronic.
 96. The method of any one of claims 74-93, wherein the method of treating is perioperative.
 97. The method of any one of claims 74-93, wherein the method of treating is intermittent.
 98. The method of any one of claims 74-97, wherein the antibody exhibits graded inhibition.
 99. The method of any one of claims 74-97, wherein the antibody exhibits switch-like inhibition.
 100. The method of any one of claims 88-99, wherein the subject suffers from two or more conditions selected from the group consisting of: bleeding disorders, platelet disorders, trauma, and bleeding resulting from a surgery or a medical procedure.
 101. The method of any one of claims 86-100, wherein the subject suffers from a bleeding disorder selected from the group consisting of: hemophilia A, hemophilia B, von Willebrand disease (vWD) disease, menorrhagia, Factor I deficiency, Factor II deficiency, Factor V deficiency, Factor VII deficiency, Factor X deficiency, Factor XI deficiency, Factor VIII deficiency (hemophilia A), Factor IX deficiency (hemophilia B), trauma, and hereditary hemorrhagic telangiectasia.
 102. The method of any one of claims 74-101, wherein the subject is a hemophilia carrier.
 103. The method of any one of claims 75-102, wherein the subject suffers from menorrhagia.
 104. The method of any one of claims 74-103, wherein the subject suffers from menorrhagia associated with one or more of the bleeding disorders or the platelet disorders.
 105. The method of claim 101, wherein the bleeding disorder is vWD, and wherein the subject is undergoing a prophylactic treatment.
 106. The method of claim 101, wherein the bleeding disorder is vWD, and the vWD is a subtype selected from: vWD Type 1, vWD Type 2A, vWD Type 2B, vWD Type 2N, vWD Type 2 M, vWD Type 3, and acquired vWD.
 107. The method of any one of claims 88-106, wherein the condition is a platelet disorder selected from the group consisting of: Bernard-Soulier syndrome, Glanzmann’s thrombasthenia, and platelet storage pool deficiency.
 108. The method of claim 107, wherein the platelet disorder is a platelet storage pool deficiency selected from the group consisting of: Gray platelet syndrome, Quebec platelet disorder, and MYH9-related thrombocytopenia (MYH9RD).
 109. The method of claim 101, wherein the bleeding disorder is selected from Factor I deficiency, Factor II deficiency, Factor V deficiency, Factor VII deficiency, Factor X deficiency, Factor XI deficiency, Factor VIII deficiency (hemophilia A), Factor IX deficiency (hemophilia B), and vWD disease, and wherein the subject has inhibitors.
 110. The method of any one of claims 88-109, wherein the antibody or the pharmaceutical composition is capable of promoting thrombin generation in the subj ect.
 111. The method of any one of claims 75-86, wherein the thrombin generation does not exceed a predetermined threshold level.
 112. The method of any one of claims 75-86 and 111, wherein the thrombin generation is antibody concentration-dependent.
 113. A kit or article of manufacture comprising an antibody of any one of claims 1-53 or the pharmaceutical composition of claim
 54. 114. Use of the antibody of any one of claims 1-53 or the pharmaceutical composition of claim 54 for the treatment of a condition in a subject in need thereof.
 115. Use of the antibody of any one of claims 1-53 or the pharmaceutical composition of claim 54 for the manufacture of a medicament for the treatment of a condition in a subject in need thereof. 